InteractiveFly: GeneBrief

Telomere fusion/ATM: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

ATM is a large, multifunctional protein kinase that regulates responses required for surviving DNA damage, including functions such as DNA repair, apoptosis, and cell cycle checkpoints. Drosophila ATM (accepted FlyBase name: Telomere fusion) function is essential for normal adult development. Extensive, inappropriate apoptosis occurs in proliferating atm mutant tissues, and in clonally derived atm mutant embryos, frequent mitotic defects are seen. At a cellular level, spontaneous telomere fusions and other chromosomal abnormalities are common in atm larval neuroblasts, suggesting a conserved and essential role for dATM in the maintenance of normal telomeres and chromosome stability. Evidence from other systems supports the idea that DNA double-strand break (DSB) repair functions of ATM kinases promote telomere maintenance by inhibition of illegitimate recombination or fusion events between the legitimate ends of chromosomes and spontaneous DSBs. Drosophila will be an excellent model system for investigating how these ATM-dependent chromosome structural maintenance functions are deployed during development. Because neurons appear to be particularly sensitive to loss of ATM in both flies and humans, this system should be particularly useful for identifying cell-specific factors that influence sensitivity to loss of dATM and are relevant for understanding the human disease, ataxia-telangiectasia (Silva, 2004).

The phosphoinositide 3-kinase-related kinases (PIKK) family in mammals includes six subfamilies based on sequence homology and function. Human ataxia-telangiectasia mutated (ATM) and ATM and Rad-3-related (ATR) proteins are the key kinases that transduce signals in response to various types of DNA damage. Drosophila mei-41 was originally reported as the ATM ortholog. However, sequence analysis reveals that CG6535, a gene predicted by the annotated Drosophila genome, is more closely related to ATM, and mei-41 actually belongs to the ATR subfamily. A related kinase ATX (CG32743 in Drosophila) plays a role in eliminating RNA species containing premature termination codons in C. elegans, whereas in humans it may have a role in the DNA damage response. Two other PIKK family members Drosophila CG2905 and CG5092 are closely related to TRRAP and mTOR, respectively, whereas the catalytic subunit of DNA-dependent protein kinase is not present in Drosophila. Among these kinases, flies with mutations in mei-41 and mTOR have been studied in detail, whereas the functions of CG2905, CG6535, and CG32743 in Drosophila remain unknown (Song, 2004 and references therein).

When a eukaryotic cell is exposed to a genotoxic treatment such as ionizing radiation that induces a DNA double-strand break, the response is led by ATM and ATR, which activate DNA repair and cell-cycle arrest in the damaged cell. ATM and ATR proteins were conserved in all eukaryotes examined. Components of the so-called MR protein complex, Rad50 and the less-well conserved Nbs1, cooperate with ATM/ATR homologs in DNA damage responses, with MR proteins activating ATM/ATR homologs and vice versa (Purdy, 2004 and references therein).

ATM/ATR homologs and MR proteins also respond to the naturally occurring DNA double-strand 'breaks'-- telomeres -- at the ends of linear chromosomes. Human, mouse and yeast cells that are deficient in ATM/ATR homologs have shorter telomeres which often fuse to other telomeres or induced double-strand breaks. Likewise, mutations in MR proteins led to shortened and fused telomeres in yeast. In these organisms, telomere length is maintained by telomerase, a reverse transcriptase which restores the GC-rich repeats at chromosome ends that would otherwise shorten after each round of DNA replication. The ends are further protected by the binding of proteins such as TRF2 homologs, and by DNA secondary structures. Genetic analyses suggest that ATM/ATR and MR proteins function to maintain telomere length or to prevent telomere fusion by telomerase-dependent and telomerase-independent mechanisms. The exact nature of these mechanisms, and their relative contribution to telomere maintenance, needed clarification, and hence, these studies (Purdy, 2004 and references therein).

Six papers have now reported that the Drosophila homologs of ATM (allelic to the previously described gene telomere fusion), Mre11 and Rad50 play a role in telomere maintenance. Mutants defective in each of these proteins show increased fusion of telomeres, with the proportion of mitotic cells showing at least one fusion event ranging from 50% to over 90%. Drosophila telomeres are unusual; they are made up of repeats of Het-A and TERT retrotransposons that are maintained by transposition rather than telomerase activity, and are protected by binding of two proteins, heterochromatin protein 1 (HP1) and HP1, ORC2 associated protein (HOAP). The new studies (Bi, 2004; Ciapponi, 2004; Oikemus, 2004; Silva, 2004; Song, 2004; Gorski, 2004) clearly define telomerase-independent contributions of ATM and MR proteins to telomere maintenance and, specifically, in the prevention of fusions. Furthermore, it now appears that the role of ATM/ATR and MR proteins in telomere protection is universal, and occurs regardless of other mechanisms that lengthen telomeres or protect them. The consequences of the loss of this protection in Drosophila are devastating; fused telomeres seem to induce a breakage-fusion cycle and lead to apoptosis and organismal death. Thus, paradoxically, DNA damage checkpoints that have been shown to promote apoptosis in response to DNA damage are fulfilling an anti-apoptotic role indirectly through telomere protection (Purdy, 2004 and references therein).

A possible mechanism for telomere protection by ATM and MR proteins in Drosophila is suggested by two findings. (1) Het-A sequences are still present at telomeres in mutants, even in those participating in fusion. Although it was difficult to quantify the extent of sequences present, it is clear that total recession of Het-A repeats is not necessary for fusion. (2) Two telomere-associated proteins, HOAP and HP1, are missing or present at reduced levels from telomeres in ATM, Rad50 and Mre11 mutants. The reduction of HOAP is apparent in mitotic cells in Mre11 and Rad50 mutants, while both HOAP and HP1 are reduced on telomeres of polytene chromosomes in ATM, Mre11 and Rad50 mutants. Because HOAP and HP1 are known to prevent telomere fusion in mitotically proliferating cells, reduction of these proteins may explain why telomeres fuse in ATM, Mre11 and Rad50 mutants. The fusions appear to be due, to a significant degree, to end-to-end joining of telomeres. Mutations in Drosophila ligase IV reduced, but did not abolish, telomere fusion in ATM mutants (Bi, 2004), similar to findings in budding yeast and mammals. Since ligase IV homologs are needed to repair a DNA double-stranded break by non-homologous end-joining, the latter may be the mechanism for at least some telomere fusion events (Purdy, 2004 and references therein).

Drosophila Mre11 and Rad50 mutants are defective in ionizing-radiation-induced repair of DNA (Ciapponi, 2004). Mre11 and Rad50 homologs also act in the repair of DNA double-strand breaks in yeast and mammals. Thus, a question posed by previous work is revisited: how can the same proteins act to repair DNA double-strand breaks induced, for example, by radiation, while acting to preserve them stably as telomeres if the ‘breaks’ are chromosomal termini? This question becomes all the more pressing because internally deleted chromosome ends, such as those generated by mobilizing of a transposable element, can recruit HOAP and HP1 to function stably as telomeres for generations in Drosophila (Fanti, 1998; Cenci, 2003). Assuming that MR proteins are also needed for recruitment of telomeric proteins to internally deleted ends, what is different about those deletions that lead to protein recruitment and telomere formation, while other deletions are repaired through the activity of the same proteins? A related question is how HP1 and HOAP are recruited to telomeres in a MR-dependent manner but not to double-strand breaks caused by ionizing radiation, for instance? Conversely, if repair enzymes are recruited to a DNA double-strand break caused by damaging agents, why are they not recruited to chromosome ends (Purdy, 2004 and references therein)?

In addition to activating DNA repair, DNA double-strand breaks also activate a checkpoint that causes cell-cycle delays. ATM/ATR and MR proteins are needed for this checkpoint in yeast and mammals. The checkpoint role of Drosophila Mre11 and Rad50 remains to be examined, but Drosophila ATM may have a minor role that is apparent at shorter times after irradiation with mutants showing robust regulation of mitosis at later times. The question then is: if double-strand breaks produce cell-cycle delay, why do not telomeres? ATM and Mre11 were recently shown to become co-localized at telomeric foci in senescent human cells. Depletion of ATM caused these cells to re-enter the cell cycle. Thus, in senescent cells, ATM appears to recognize telomeres as DNA breaks and cause cell-cycle arrest. An intriguing possibility, then, is that onset of senescence may simply reflect a switch in which ATM goes from having a telomere-protective role that does not result in cell-cycle arrest, to a DNA-damage checkpoint role that does result in cell-cycle arrest and hence senesence. In any case, addressing how the same set of molecules, ATM and MR proteins, could initiate different sets of outcomes, depending upon where on the chromosome a 'break' forms, should prove to be challenging and rewarding (Purdy, 2004 and references therein).

REGULATION

TCTP directly regulates ATM activity to control genome stability and organ development in Drosophila melanogaster

Translationally controlled tumour protein (TCTP) is implicated in growth regulation and cancer. Recently, human TCTP has been suggested to play a role in the DNA damage response by forming a complex with ataxia telangiectasia-mutated (ATM) kinase. However, the exact nature of this interaction and its roles in vivo remained unclear. This study utilized Drosophila as an animal model to study the nuclear function of Drosophila TCTP (dTCTP). dTCTP mutants show increased radiation sensitivity during development as well as strong genetic interaction with dATM mutations, resulting in severe defects in developmental timing, organ size and chromosome stability. Drosophila ATM (dATM) was identified as a direct binding partner of dTCTP, and a mechanistic basis is described for dATM activation by dTCTP. Altogether, this study provides the first in vivo evidence for direct modulation of dATM activity by dTCTP in the control of genome stability and organ development (Hong, 2013).

TCTP has been implicated in a few events in cell nucleus, including transcription. Recently, it has been reported that hTCTP in irradiated fibroblast participates in DSB repair through Ku translocation and colocalization with ATM. However, the nature of hTCTP interaction with ATM and its physiological significance in animals remains unknown (Hong, 2013).

These in vivo studies, using Drosophila as an animal model, provide evidence that dTCTP is required for maintaining genome stability in mitotic cells of developing organs such as brain and wing discs, and endocycling cells of salivary glands. In response to induced DSBs, dTCTP mutants show reduced viability, abnormal array of ommatidia, increased DNA fragmentation, radioresistant DNA synthesis (RDS; intra S checkpoint defect), G2/M checkpoint defect and increased apoptosis as a result of impaired checkpoints. Since these defects are similar in Drosophila atm mutants, it is likely that the function of dTCTP for DSB repair is closely related with dATM. Indeed, both direct interaction and subcellular colocalization between them support this notion. Moreover, genetic data and biochemical assays using dATM and its substrate (H2Av) illustrate that dTCTP directly facilitates dATM kinase activity by enhancing binding affinity of dATM to its substrate, providing a molecular basis for the relationship between TCTP and ATM that has not been studied before. It is interesting to note that dATM is required for the formation of dTCTP foci but not vice versa. Hence, in the first place dTCTP may be recruited to nuclear foci depending on the presence of dATM. Once it is recruited to the DSB sites, dTCTP may function as a cofactor for positive modulation of dATM kinase activity, providing a mutually dependent mechanism for dATM activation (Hong, 2013).

DSBs are repaired by one of the two major repair pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways. In HR, the activated ATM phosphorylates many proteins to facilitate DNA damage repair. The Mre11-Rad50-Nbs1 (MRN) complex (a DSB sensor) is also required to activate and recruit ATM to DSB sites in HR pathway. In NHEJ, DSBs are sensed by DNA-PKcs/Ku70/80 protein complex (DNA-PK holoenzyme) and repaired by Lig4. Interestingly, a subset of HR-associated proteins such as MRN and ATM can interlink HR and NHEJ. For example, the MRN complex is partially involved in DSB end processing for NHEJ repair. ATM also enhances the NHEJ process by phosphorylating the nuclease Artemis for DSB end processing as well as Ku70/80 complex to enhance its binding affinity to DNA fragments (Hong, 2013).

Genetic data from this study show strong interactions between dTCTP and DNA damage response (DDR) genes for HR (MRN, MDC, MCPH1 and his2Av) and NHEJ (MRN, mus309 (a.k.a. ku70 homologue and Bloom syndrome helicase ortholog)). These results suggest that dTCTP can interlink HR and NHEJ in vivo. This idea is also consistent with a model that hTCTP interlinks the HR and NHEJ pathways. In this model, hTCTP promotes Ku70/80 complex translocation for NHEJ repair and forms a complex with ATM for HR repair, although its function in HR pathway has not been clearly defined. Collectively, these studies in human and fly strongly support that TCTP participates in both HR and NHEJ pathways to repair DSBs in eukaryotes (Hong, 2013).

In addition to the fact that both Drosophila and hTCTP are involved in HR and NHEJ repair pathways, they have several other features in common. Reduced TCTP function results in increased chromosomal fragmentation by IR, IR sensitivity, RDS (G1/S-phase checkpoint defect) and G2 delay (G2/M checkpoint defect). TCTP also forms a complex with ATM in DSB sites and participate in NHEJ process for DNA repair by helping Ku translocation (Hong, 2013).

However, there are also apparent differences between hTCTP and dTCTP. Unlike hTCTP, dTCTP protein level or stability is not affected by dATM or IR. Importantly, dTCTP localization in the endocycling cells suggests that dTCTP is needed to repair not only induced DSBs but also naturally occurring DSBs in physiological condition. One of the most remarkable differences is shown in the phosphorylation level of H2A variant (γ-H2Av in fly and γ-H2AX in human) in the absence of TCTP. In irradiated human fibroblasts, hTCTP depletion causes increases in DNA fragmentation and H2AX phosphorylation compared with the normal control. In contrast, reduction of dTCTP in dTCTP mutants leads to increased DNA fragmentation but reduced H2Av phosphorylation levels. Previous reports in Drosophila adult brain, mouse embryonic fibroblast and human osteoblastoma have shown that H2Av/H2AX phosphorylation levels are considerably reduced in ATM-depleted conditions despite high DNA lesions, and the current results are on the same lines as these. Thus, the current data support the hypothesis that TCTP directly enhances ATM kinase activity to repair DSBs, especially in the early DDR steps such as amplification of DSB signals through γ-H2AX formation by ATM. in vitro assay data strongly support this concept (Hong, 2013).

An intriguing question is how H2AX phosphorylation is increased in hTCTP-depleted fibroblasts, on the contrary to dTCTP- and ATM-depleted conditions. One possible explanation is that γ-H2AX might be formed by redundant functions of other kinases such as ATR or DNA-PK that can substitute the reduced ATM activity resulting from TCTP depletion. Such compensatory phosphorylation might be possible because both ATR and DNA-PK can phosphorylate H2AX to some extent against induced DSBs when ATM is depleted. On the contrary, Drosophila atm⁸ mutant shows little H2Av phosphorylation in response to induced DSBs. Furthermore, Drosophila atr mutants show weak genetic interactions with ey>dTCTPi resulting in mildly enhanced eye phenotypes. These may indicate that functional redundancy for dATM is restricted and that the mechanism to maintain genome stability in Drosophila is simpler than in human. It would be interesting to see whether TCTP proteins in other vertebrate systems function to control ATM activity as in Drosophila (Hong, 2013).

Growing evidence suggests that mammalian ATMs are known to play additional roles in the cytoplasm, although the underlying mechanisms are not well understood. Interestingly, recent studies suggested that human ATM regulates mTORC1 pathway either positively or negatively depending on the presence or absence of stresses. Given that TCTP makes a complex with ATM and functions in both nucleus and cytoplasm, it could be possible that TCTP and ATM might share a function for modulating mTORC1 pathway (Hong, 2013).

DEVELOPMENTAL BIOLOGY

To compare the in vivo functions of dATM and mei-41, their developmental expression patterns were examined. The dATM gene is expressed at relatively low levels throughout development and at much higher levels in adult females, when compared to males. Similarly, mei-41 is expressed at high levels in adult females and ovaries and is required for meiosis, suggesting a potential role for both genes in the female germ line (Song, 2004).

EFFECTS OF MUTATION

Telomeres are the stable ends of linear chromosomes in eukaryotes. These complex protein-nucleic acid structures are essential to maintain genomic stability and the integrity of linear chromosomes. A new mutation (tef for telomere fusion) was identified in Drosophila that causes a high frequency of end-to-end fusions of chromosomes during mitosis and meiosis. Linear chromosomal ends appear to be essential for fusions to take place. These fusions do not resolve, leading to cycles of chromosomal breakage and rejoining and severe genome rearrangements. The gene is essential for normal cell proliferation and mutant tissue shows significant apoptosis. This analysis suggests that the function encoded by the mutant gene is required to protect the linear ends of chromosomes (Queiroz-Machado, 2001).

ATM is required for telomere maintenance and chromosome stability during Drosophila development

To determine the function of atm, a genetic screen was carried out that isolated eight recessive lethal alleles. Seven of these alleles (atm^1-7) have a nonconditional, pupal, lethal phenotype and one, atm⁸, is a temperature-sensitive hypomorphic allele. Hemizygous atm^1-7 mutants die before emerging as adults, with variable morphological defects affecting the antennae, eyes, bristles, wings, and legs. The completely restrictive temperature for atm⁸ lethality is approximately 25°C. When atm⁸ mutants are raised at a semirestrictive temperature (24°C± 1°C), many can survive to adulthood with a similar range of morphological defects. The morphological defects, as well as atm mutant lethality, are rescued by introduction of a transgenic construct, P{atm⁺}, confirming that these phenotypes are specifically due to loss of atm function (Silva, 2004).

To determine when dATM activity is required for normal development, advantage was taken of the temperature-sensitive allele atm⁸ in a reciprocal temperature-shift experiment that established the temperature-sensitive period (TSP) for selected atm⁸ mutant phenotypes. The TSP for adult viability falls between the early third-larval instar and early pupal stage, the TSP for eye development is during the third-larval instar, and the TSP for wing and thoracic bristle development is during the pupal stage. These TSPs correspond to critical developmental stages of eye, wing, and bristle development. Requirements for dATM activity appear particularly stringent in developing neural tissues, because the structures most obviously affected in the atm mutants (eye, wing margin, thoracic bristles) are all involved in sensory perception (Silva, 2004).

Because movement disorders (ataxia) are a prominent symptom in patients with A-T, whether atm⁸ mutants exhibit signs of locomotor defects was examined. Climbing ability was examined in atm⁸ mutant and control flies for this purpose. Replicate tests were conducted for each fly, and the averages were used for deriving a ratio (mutant/control) that provided a measure of how well atm⁸ mutants climb, relative to matched controls. The average climbing ability of atm⁸ mutant males was markedly lower than normal (ratio of mutants/controls = 0.11). Interestingly, the atm⁸ mutant females performed even worse in this assay (mutants/controls = 0.01). The relative differences in climbing ability between mutants and controls did not change over a period of 4 weeks when the flies were maintained at the restrictive temperature of 29°C. Thus, the locomotor defects in atm mutants appear to be strictly developmental (Silva, 2004).

Because the temperature-sensitive period for normal eye development in atm⁸ mutants is during the third-larval instar, eye-antennal discs were examined at this stage of development. A structure called the morphogenetic furrow (MF) sweeps across the eye disc during the late-third instar, marking a transition between proliferating, undifferentiated cells (anterior to the furrow) and nonproliferating cells that are differentiating into distinct neuronal cell types, posterior to the furrow. The atm⁸ mutant-eye antennal discs are generally smaller than comparably aged controls. Conspicuously, atm⁸ mutant eye discs had large numbers of cells undergoing inappropriate apoptosis, marked by antibody staining for activated caspase-3. There were striking regional differences in the distribution of apoptotic cells in the mutant eye discs; these differences were also seen in discs stained with acridine orange staining as another marker of apoptosis. These apoptotic cells were primarily restricted to the anterior, proliferating region of the disc. There were relatively few apoptotic cells in the posterior region of the mutant discs, containing differentiating neuronal cells. Early neuronal patterning was markedly disrupted in mutant discs, presumably because so many cells were eliminated by earlier apoptosis that normal precluster formation was precluded (Silva, 2004).

The adult wing is also affected by loss of atm function, so atm⁸ mutant third-larval instar wing discs were examined for developmental defects. At this stage, cells in the wing disc are proliferating asynchronously except for a stripe of nondividing, differentiating cells along the presumptive wing margin of the disc. These cells express Cut, a Notch-induced transcription factor required for proper differentiation of the wing margin. A partial to complete loss of Cut expression was observed along the presumptive wing margin of atm⁸ mutant discs, a patterning defect that anticipates the wing margin defects seen in atm mutants later in development. Intriguingly, Cut expression was not obviously affected in the myoblast cells adjoining the presumptive dorsal thorax region of the atm mutant wing discs. The Cut expression defect in the developing wing margin could be due to inappropriate cell death in the proliferating imaginal cells, because spontaneous apoptosis also occurs throughout the atm⁸ mutant wing discs at this developmental stage. The spontaneous apoptosis and patterning defects in atm eye and wing imaginal discs suggest that cells are experiencing DNA damage, which is an established trigger for p53-dependent apoptosis in Drosophila (Silva, 2004).

Oogenesis and early embryogenesis are regulated by maternally acting genes in Drosophila. To determine if dATM activity is required during early stages of development, eggs derived from atm⁶ mutant-germline mosaic females, generated using the hs-FLP/FRT ovo^D system, were examined. atm⁶ was molecularly characterized as a functional null allele. Maternal mutant atm⁶ eggs commonly have dorsal appendage defects, resembling a phenotype of homologous DSB repair-defective female-sterile mutants. The eggshells of atm⁶ mutant eggs also appear to be thinner than normal, suggesting that follicle cell development is compromised. Developing atm⁶ mutant embryos show dramatic mitotic defects during the rapid syncytial divisions of early embryogenesis. These mitotic defects include frequent spindle fusions and chromosomal bridges that could also be observed much earlier in development before the nuclei reach the surface of the embryo. Another frequently observed defect involved nuclei detaching from centrosomes and falling from the cortex (Silva, 2004).

The centrosome and nuclear fallout defect in atm mutants resembles a local DNA damage-induced response called 'centrosome inactivation', indicating that loss of dATM is associated with spontaneous DNA damage. Chk2 is an established transducer of ATM signaling in mammalian cells and Loki, the Drosophila Chk2 homolog, has been identified as a positive regulator of centrosome inactivation in Drosophila. Because centrosome inactivation occurs spontaneously in atm mutants, this result suggests that either a dATM-independent mechanism for activating Chk2 exists or a Chk2-independent mechanism for promoting centrosome inactivation was deployed (Silva, 2004).

In mammalian cells, the ATM and ATR signaling pathways negatively regulate Cdk1 in response to ionizing radiation, blocking mitosis while DNA is being repaired. Mei-41, the Drosophila ATR ortholog, is required for this response to DNA damage. To examine if dATM might also be required for this response, mitotic cells were labeled in atm mutant and control wing discs, with or without exposure to 40 Gy of γ irradiation. Mitotic cells were essentially absent 1 hr after ionizing radiation in both atm mutant and control discs, implying that dATM is dispensable for this premitotic-checkpoint response to acute DNA damage. dATM operates in a premitotic checkpoint, which is activated earlier in response to ionizing radiation than the measurements carried out in this study but the checkpoint response is fully engaged by 1 hr after irradiation (Silva, 2004 and references therein).

Increased numbers of apoptotic cells were observed in both controls and atm mutant wing discs, after larvae were exposed to ionizing radiation. Because the apoptosis response to ionizing radiation in Drosophila is thought to depend on p53, this result implies that p53-dependent apoptosis is functional in atm mutants. Indeed, expression of a dominant-negative p53 transgene suppresses spontaneous apoptosis in atm mutant wing discs, supporting the conclusion that the p53-dependent apoptosis response is intact (Silva, 2004).

atm mutants are extremely sensitive to ionizing radiation. Radiation sensitivity was assayed by comparing the adult viability of atm⁸ mutants, raised at either semirestrictive (24°C) or permissive (22°C) temperatures, after third-instar larvae were exposed to different doses of γ radiation. After taking into account the loss of adult viability caused by raising the mutants at 24°C, the data show that atm mutant viability is drastically compromised by exposure to ionizing radiation, even at the lowest dose of 1 Gy. Thus, it is concluded that dATM is required for cells to survive both spontaneous and induced DNA damage (Silva, 2004).

ATM has been implicated in two processes that are fundamental to chromosome integrity: repair of DNA double-strand breaks (DSBs) and telomere maintenance. To examine chromosome stability, larval neuroblasts from atm mutant and control larvae were examined; an astonishingly high rate of spontaneous chromosomal aberrations was observed in the mutants. The most frequent aberrations were telomere fusions, affecting >50% of metaphase spreads. Both single- and double-telomere associations (TA) were observed, including sister and nonsister fusions of homologous chromosomes, as well as fusions between nonhomologous chromosomes. Acentric chromosome fragments were also observed on rare occasions, and chromosomal transpositions involving the loss of whole chromosome arms. Dicentric chromosomes resulting from telomere fusions are inherently unstable, because the kinetochores can be captured by microtubules from opposite spindle poles, causing chromosome bridges that eventually rupture, triggering apoptosis. Consistent with this expectation, frequent chromosomal bridges were observed in atm mutant anaphase spreads. Collectively, these data implicate dATM in normal telomere maintenance and chromosome stability (Silva, 2004).

These results indicate that the primary function of dATM is in chromosome structural maintenance, without which proliferating cells are vulnerable to apoptosis. dATM is also implicated in an early premitotic checkpoint response to ionizing radiation, suggesting that ATM and ATR carry out temporally distinct cell cycle checkpoint functions as they do in mammals. Also in mammals, the DNA repair functions of ATM overlap with those of a functionally redundant, ATM-related kinase called DNA-PK. DNA-PK is not conserved in Drosophila, perhaps explaining why ATM is essential for morphological development in Drosophila but not in mammals (Silva, 2004).

Inappropriate apoptosis occurs predominantly in proliferating atm mutant cells, suggesting that dATM is required for repairing spontaneous DSBs arising during DNA replication. The extraordinary frequency of spontaneous telomere fusions in atm mutant neuroblasts also suggests a critical role for dATM in telomere maintenance in cycling cells. Drosophila telomeres are unusual because they are maintained by retrotransposition rather than by telomerase activity, as they are in other systems. Given this difference, it is very intriguing that ATM has now been linked to telomere maintenance in eukaryotic organisms as distantly related as yeast, Drosophila, and humans. The molecular mechanisms of ATM-dependent telomere maintenance have not yet been established in any organism, however, studies in yeast suggest that ATM/TEL1 activity prevents spontaneous chromosomal rearrangements involving telomeres during the repair of DSBs. The conserved MRN protein complex (comprised of Mre11, Rad50, and Nbs1) is required for all known ATM-dependent functions including telomere maintenance. Mutations affecting Drosophila mre11 and rad50 also result in pupal lethality and telomere fusions, consistent with these genes being involved in common DNA repair and chromosome structural maintenance functions. Recent genetic screens have identified a number of Drosophila genes of unknown function that are required for preventing spontaneous telomere fusions, many of which are probably also involved in ATM-dependent telomere maintenance functions (Silva, 2004 and references therein).

Drosophila atm mutants recapitulate major symptoms of ataxia telangiectasia including locomotor defects, sensitivity to ionizing radiation, and chromosome instability. Curiously, loss of ATM seems to affect developing neuronal tissues more than non-neuronal tissues, in both flies and humans. This correlation may reflect a relationship between telomere dysfunction and cell proliferation. Alternatively, specific developmental events may render certain cells more sensitive to loss of ATM function. Chromatin remodelling has been shown to activate ATM in cultured mammalian cells, making it tempting to speculate that neuron-specific chromatin remodelling processes might normally elicit ATM activity. Drosophila promises to be an excellent model for investigating the basic mechanisms of chromosome structural maintenance involving ATM, allowing these possibilities to be studied in a meaningful developmental context (Silva, 2004 and references therein).

Telomere protection without a telomerase: The role of ATM and Mre11 in Drosophila telomere maintenance

The conserved ATM checkpoint kinase and the Mre11 DNA repair complex play essential and overlapping roles in maintaining genomic integrity. Genetic and cytological studies were conducted on Drosophila atm and mre11 knockout mutants. A telomere defect was discovered that was more severe than in any of the non-Drosophila systems studied. In mutant mitotic cells, an average of 30% of the chromosome ends engaged in telomere fusions. These fusions led to the formation and sometimes breakage of dicentric chromosomes, thus starting a devastating breakage-fusion-bridge cycle. Some of the fusions depended on DNA ligase IV, which suggests that they occurred by a nonhomologous end-joining (NHEJ) mechanism. Epistasis analyses results suggest that ATM and Mre11 might also act in the same telomere maintenance pathway in metazoans. Since Drosophila telomeres are not added by a telomerase, these findings support an additional role for both ATM and Mre11 in telomere maintenance that is independent of telomerase regulation (Bi, 2004).

The ATM checkpoint kinase and the Mre11 DNA repair protein function to maintain telomere integrity in yeast and mammals cells (reviewed in Ferreira, 2004). Whether or not they have a similar role in Drosophila, an organism that lacks a canonical telomerase, was investigated. Both atm and mre11 were knocked out by targeted mutagenesis. For atm, two different alleles were recovered by ends-in gene targeting, referred to here as atm^strong (atm^stg) and atm^weak (atm^wk). For mre11, a complete knockout was achieved by deleting the entire Mre11 coding region by using ends-out gene targeting. Both atm and mre11 mutant animals were late pupal lethal. Proliferating tissues in the mutants experienced an excess amount of cell death. Both atm alleles failed to complement a chromosomal deficiency (Df(3R)hsc70-4^Δ356) that deleted part of atm and the adjacent hsc70-4 gene. In addition, animals were generated that were homozygous for the deficiency and a wild-type hsc70-4⁺ transgene, thus rescuing the Hsc70-4 function. These animals were pupal lethal and displayed the same defects as atm knockout homozygotes. The pupal lethality of the mre11 deletion mutant was fully rescued by a wild-type mre11⁺ transgene (Bi, 2004).

To pinpoint the underlying genetic causes for the massive cell death, DAPI-stained mitotic chromosomes of neuroblasts were examined from the brains of third instar larvae. The most prominent cytological defect that was observed was chromosome end-to-end association (telomere attachment [TA]). A TA can involve any of the four chromosome pairs. TA was categorize into three different classes: single TA, double TA, and others. A single TA most likely occurred during S/G₂ after telomeric DNA replication had completed, whereas a double TA could be derived from a single TA in G₁ by replication. Besides widespread end-to-end attachments, severe genome instability was also observed in the forms of chromosome breakage, chromosome rearrangements, and gross aneuploidy. Chromosome breakage is grouped into two types: chromatid breaks and chromosome breaks. For example, a chromatid break was observed that involved only one of the two sisters. As a second example, an observed chromosome break involved both sister chromatids broken at an identical region and was likely the result of the replication of a single G₁ break; such a G₁ break could occur de novo. It could also occur as a result of a broken dicentric chromosome during the previous cell division. Interestingly, not only could the natural chromosome ends associate with each other in the mutants, they could also do so with broken ends, thus creating some of the chromosome rearrangements, which include nonreciprocal translocations, and terminal deficiencies. In addition, polyploid nuclei were observed undergoing chromosome condensation (Bi, 2004).

Consistent with atm^wk being a weaker allele, an average atm^wk/wk cell had about one fewer TA than an atm^stg/stg cell. In fact, over 20% of the atm^wk/wk nuclei had no TA, compared to 6% for atm^stg/stg. An average atm^wk/wk cell also had lower frequencies for both chromosome breakage and aneuploidy than an atm^stg/stg cell. Cells from atm^stg/stg and mre11^-/- had similar numbers of TAs on average, which was similar to the rate in atm^stg/Df(3R)hsc70-4^Δ356 cells. In contrast, mre11^-/- cells possessed more breaks and were more likely to be aneuploid than atm^stg/stg cells. This excess of genome instability in mre11^-/- cells was likely due to additional repair defects since Mre11 participates in multiple processes in DNA recombination and repair. In order to establish epistasis for ATM and Mre11 in telomere maintenance, mre11^- and atm^stg double homozygotes were generated by crossing. At the chromosomal level, a double mutant nucleus had a number of TAs similar to atm^stg/stg but slightly more than mre11^-/-. This lack of an additive effect from the double mutations led to the conclusion that ATM and Mre11 were in the same pathway for telomere protection, similar to their epistasis relationship in yeast (Ritchie, 2000). At the organismal level, double mutant animals died as late third instar larvae, which was a few days earlier than either of the singles. This earlier death suggested nonoverlapping functions for either protein. Indeed, the double mutation had an additive effect on the frequency of breaks, suggesting that the two proteins might contribute in parallel to prevent chromosome breakage (Bi, 2004).

In certain yeast double mutants involving tel1 (yeast atm) or mre11, telomere fusions were preceded by extensive telomeric DNA loss (Chan, 2003; Mieczkowski, 2003). Attempts were made to determine whether that also applied to Drosophila mutants. In none of the mutant combinations was a reduction observed of the overall abundance of the terminal HeT-A elements, which are the most abundant retro-transposable elements that make up the normal ends of Drosophila chromosomes (reviewed in Pardue, 2003). However, since the short telomeres in tel1 or mre11 yeast mutants were not reached until after about 80 cell divisions (Ritchie, 2000), it is possible that only cells undergoing mitosis would experience loss of telomeric DNA as a result of the Drosophila atm mutations. To address this issue, in situ hybridization was carried out on mitotic chromosomes. Telomeric HeT-A signals were often observed at the point where two chromosomes fused. This led to the conclusion that at least some of the fusions occurred without complete loss of telomeric DNA. Since fusions were also observed without HeT-A signals, it could not be ruled out that some of the fusions were preceded by DNA loss (Bi, 2004).

Telomere fusions can lead to the formation of dicentric bridges during anaphase of mitosis. For both mutants, bridges were observed in chromosome squashes of larval neuroblasts, but at a low frequency, possibly due to hypotonic treatment of the cell prior to squashing. To gain a more comprehensive understanding of the consequences that follow mitotic bridge formation, chromatin was stained with an antibody against a phosphorylated form of histone H3 specific to mitosis. This enabled the observation to be made that mitotic bridges were frequently observed in cells from all the proliferating tissues studied; this suggested that the telomere protection function of ATM and Mre11 was required for all proliferating tissues. On average, about 20% of the mitotic nuclei from the brain and imaginal discs displayed abnormal chromosome configurations. A few interesting points were noted: (1) multiple bridges were commonly observed, consistent with the mutant nucleus having multiple TAs; (2) bridges could persist without breakage, at least until telophase (they might be eventually severed by cytokinesis); (3) acentric fragments were observed left at the metaphase plate, most likely leading to aneuploidy; (4) broken bridges were frequently observed. Bridges were most often broken around the midpoint. However, asymmetrically broken bridges were also present, which would lead to a nonreciprocal translocation. Some bridges showed multiple constrictions indicative of chromatin breaks that might not involve DNA. This provides physical evidence supporting the idea that breakage of mitotic bridges is a feasible mechanism for the generation of internal breaks and supports the hypothesis that most of the gross chromosome rearrangements seen in certain yeast repair mutants arise as a result of a breakage-fusion-bridge cycle (Bi, 2004).

In both yeast and mammalian cells, covalent telomere fusions in certain mutant backgrounds often occur by NHEJ that require the specialized DNA ligase IV (reviewed in Ferreira, 2004). To test the requirement for ligase IV, mutations were generated in the Drosophila ligase IV (lig4) gene by mobilizing a nearby P element (EP0385); a mutation was recovered that deleted the first 147 codons (Bi, 2004).

At the chromosomal level, a lig4^- atm^stg nucleus had slightly fewer TA on average than an atm^stg single mutant. This lack of a larger effect was verified with another lig4 deletion mutation. For the atm^wk mutation, loss of Lig4 function significantly reduced the overall TA rate. Interestingly, different classes of TA responded differently to the mutation. The rate for double TAs was most sensitive, dropping from 0.98 to 0.46. This suggested that Lig4 was more important for attachments that occurred in G₁ in which NHEJ activity predominates for both yeast, mammals, and perhaps, Drosophila. In contrast, the rate for intersister TAs was not at all affected. Since sisters are identical in sequence, an intersister TA could occur by a homologous recombination-based mechanism, which does not require Lig4. It is concluded that at least some of the TAs in atm mutants involved covalent joining of telomeric DNA. This was also supported by the behaviors of mitotic bridges described earlier. To explain the allele-specific response to the lig4 mutation, it is first suggested that NHEJ is not entirely dependent on Lig4 in Drosophila. It is then imagined that in an atm^stg/stg cell, the half-life in which a telomere could participate in fusions is significantly longer due to the complete loss of ATM. A fusion prevented by the lig4 mutation would be carried out by other mechanisms, which would result in a somewhat constant overall TA frequency. Based on this model, it is predicted that some of the NHEJ-typed fusions in lig4^-/- atm^stg/stg cells would be channeled into fusions of other types, perhaps one that was based on sequence homology. Consistent with this, an increase was observed of over 20% for the rate of intersister TAs accompanied by a reduction of the rate for double TAs (Bi, 2004).

The association of human Mre11 with TRF2, a telomeric repeat binding protein, suggests that Mre11 may be a structural component of a normal telomere. However, Mre11 was not localized to telomeres in a mixed population of wild-type yeast cells. Therefore, it remains possible that both ATM and Mre11 regulate the telomere protection activity of other proteins. In an effort to identify such targets, focus was placed on the Drosophila HP1/ORC-associated protein (HOAP). HOAP is the only Drosophila protein that has been shown to exclusively localize to the ends of mitotic chromosomes (Cenci, 2003). A mutation in HOAP caused telomere attachments. In addition, the small HOAP protein (337 aa) contains six S/TQ sites, which are the preferred phosphorylation sites in known targets of ATM (reviewed in Abraham, 2001). It is attractive to postulate that ATM exerts its telomere protection function by regulating HOAP localization to the telomeres and that Mre11 may do so by regulating the kinase activity of ATM (Bi, 2004).

To test this model, immunolocalization was performed of HOAP in mitotic cells from the larval brains. In either atm^stg or atm^wk mutant cells, HOAP was localized to the end normally. This normal localization was also observed in atm^stg/Df(3R)hsc70-4^Δ356 cells. In addition, HOAP could be detected on 15% of the telomeres engaged in attachments. This is identical to the results from HOAP localization studies in fly mutants lacking HP1. Therefore, HOAP localization to mitotic telomeres did not depend on ATM. However, this localization was affected by the mre11 mutation. The overall chromosomal staining of HOAP was reduced in the mre11 mutant background, with only 40% of the nuclei displaying a HOAP signal on more than one chromosomes. In these nuclei, HOAP was often missing from majority of the chromosome ends. HOAP could still be localized to telomeres engaged in TAs. Another study also discovered telomere fusion and reduced HOAP localization in another mre11 mutation (Ciapponi, 2004). HOAP localization was also studied in the mre11 atm^stg double mutant and an extent of HOAP localization similar to mre11 alone was observed. These results, particularly the ability to detect HOAP at some of the fusion junctions in both mutants, led to the conclusion that failure to recruit HOAP to the telomeres could not have been the cause for fusion. However, it cannot be ruled out that HOAP's telomere-protecting function, but not its telomere localization, still depends on ATM (Bi, 2004).

The above results are consistent with the hypothesis that Mre11 might act at or around the telomere. It is imagined that the Mre11 nuclease processes the telomere into a special structure, which facilitates HOAP binding. Absent this structure, the efficiency of HOAP binding is reduced, and the fixative treatment in the immunostaining procedure might result in variable loss of telomere bound HOAP proteins. Based on the lack of effect of the atm mutations on HOAP localization, it is proposed that ATM is not an integral part of a telomere. Instead, it may exert its regulatory role as a kinase (Bi, 2004).

Not only are Drosophila telomeres not added by a telomerase, but it was also believed that the end of a Drosophila chromosome could consist of essentially any sequence. Therefore, Drosophila telomeres, unlike ones in yeast or mammals, lack the protection conferred by the canonical telomerase and various telomeric repeat binding proteins. This would render Drosophila telomeres more vulnerable to uncapping and make the telomere protection function of ATM and Mre11 more critical for cell survival (Bi, 2004).

The mechanisms by which ATM and Mre11 maintain telomere homeostasis are largely unclear. In the most advanced model from yeast, they recruit telomerase activity to the chromosome ends by altering the structure of the telomeric DNA (Chan, 2001; Tsukamoto, 2001). A more direct role of ATM was recently suggested that was based on a synergistic effect of an atm knockout with a telomerase knockout in causing telomere dysfunction in both yeast and mouse (Chan, 2003; Qi, 2003). This study provides evidence supporting such a hypothesis, and shows that Drosophila would be an excellent system for advancing such studies without the complication of telomerase (Bi, 2004).

Drosophila ATM mediates the response to ionizing radiation and to spontaneous DNA damage during development

Cells of metazoan organisms respond to DNA damage by arresting their cell cycle to repair DNA, or they undergo apoptosis. Two protein kinases, ataxia-telangiectasia mutated (ATM) and ATM and Rad-3 related (ATR), are sensors for DNA damage. In humans, ATM is mutated in patients with ataxia-telangiectasia (A-T), resulting in hypersensitivity to ionizing radiation (IR) and increased cancer susceptibility. Cells from A-T patients exhibit chromosome aberrations and excessive spontaneous apoptosis. Drosophila has been used as a model system to study ATM function. Previous studies suggest that mei-41 corresponds to ATM in Drosophila; however, it appears that mei-41 is probably the ATR ortholog. Unlike mei-41 mutants, flies deficient for the true ATM ortholog die as pupae or eclose with eye and wing abnormalities. Developing larval discs exhibit substantially increased spontaneous chromosomal telomere fusions and p53-dependent apoptosis. These developmental phenotypes are unique to Drosophila ATM, and both Drosophila ATM and mei-41 have temporally distinct roles in G2 arrest after IR. Thus, ATM and ATR orthologs are required for different functions in Drosophila; the developmental defects resulting from absence of Drosophila ATM suggest an important role in mediating a protective checkpoint against DNA damage arising during normal cell proliferation and differentiation (Song, 2004).

Drosophila lines were generated with specific disruptions of the dATM gene. A previously generated P element insertion, EP(3)0859, located within the hsc70-4 (hsc4) gene, which is 546 bp upstream of dATM, disrupts hsc4 and exhibits homozygous lethality. In efforts made by others to generate a null allele of the hsc4 gene, this transposable element was mobilized, resulting in two different imprecise excision deletions of hsc4 including the dATM gene. These are referred to as Δ11 and Δ356. The endpoints of the deleted region were confirmed by PCR followed by sequence analysis. dATM was predicted to contain 24 exons: Δ11 flies lack the first 2 exons, whereas the first 8 exons are deleted in Δ356 flies. The flies were extensively out-crossed to wild-type flies to eliminate any additional mutations on the chromosome and then used for further analysis. The lethality resulting from the hsc4 mutation was rescued transgenetically with a 14 kb genomic fragment encompassing the complete hsc4 gene, thereby generating 'clean' loss-of-function alleles of dATM (Song, 2004).

The majority of flies with the genotype P[hsc4]; Δ11 and P[hsc4]; Δ356 exhibit pupal lethality. However, 12% (Δ11) and 15% (Δ356) of homozygous mutants eclosed and were viable, although they did not survive very long. Northern blot analysis of the 'escaper' females with the genotype P[hsc4]/Cyo; Δ11, P[hsc4]/Cyo; Δ356, and P[hsc4]/Cyo; EP(3)0859 revealed that the dATM transcript is substantially reduced in both Δ11 and Δ356 mutant flies, whereas it is present in EP(3)0859. Interestingly, Δ11/Δ356 transheterozygotes exhibit a higher percentage of viability, suggesting that there may be some complementation between the two alleles. Moreover, two additional dATM alleles that exhibit homozygous lethality were subsequently obtained from the Exelixis transposon disruption collection. These findings suggest that the Δ11 and Δ356 alleles are strong hypomorphic loss-of-function dATM alleles (Song, 2004).

dATM, mei-41, grp, and dChk2 are close orthologs of mammalian DNA damage checkpoint genes that are all highly expressed in Drosophila females relative to males. The encoded checkpoint protein kinases presumably respond to DNA double-strand breaks generated during meiotic recombination. Notably, meiotic recombination in Drosophila occurs only in females. Moreover, mammalian ATM and ATR localize to synapsed and unsynapsed regions of meiotic chromosomes, respectively, suggesting a role for these proteins during meiotic recombination. In Drosophila, most mei-41 alleles are recessive female sterile, and females homozygous for the more fertile hypomorphic mei-41 alleles exhibit reduced meiotic exchange. The Δ356 dATM mutant females do not lay eggs, whereas Δ11 females lay a very small number of eggs that do not hatch, indicating that dATM is required for female fertility. In mouse models, ATR is an essential gene, whereas ATM is dispensable for normal development and viability, although homozygous mutant mice exhibit immune defects, growth retardation, and sterility. In Drosophila, the mei-41(ATR) mutant was found to be maternal lethal and nullizygous dATM mutants were found to be lethal. Thus, unlike its mammalian counterpart, Drosophila ATM plays a critical developmental role (Song, 2004).

To determine the basis for these phenotypes, third-instar wing discs or eye discs were isolated and examined for morphology and for apoptosis by TUNEL staining. Although wing and eye disc morphology appears to be normal, dATM mutant discs are somewhat reduced in overall size and exhibit a substantial increase in TUNEL-positive cells with relatively few apoptotic cells detected within wild-type discs. To determine whether the excessive apoptosis is p53-dependent, a dominant-negative (dn) form of Dmp53 was introduced. The dn-Dmp53 transgene has been shown to block IR-induced apoptosis when expressed in the posterior part of the wing with engrailed-GAL4. Expression of dn-Dmp53 in dATM mutant wing discs results in a substantial decrease in apoptosis. Thus, loss of dATM function results in p53-mediated apoptosis during development. In a report by Bi (2004), spontaneous apoptosis in dATM mutant discs was found to occur even in a p53-zygotic mutant background, suggesting that the observed apoptosis is p53 independent. This apparent discrepancy in findings may reflect the presence of maternal Dmp53 product in larvae of the homozygous mutant background (Song, 2004).

In imaginal eye discs double-stained for TUNEL and the neuronal marker Elav, it was observed that the excessive spontaneous apoptosis seen in dATM mutants is largely restricted to the proliferating cells anterior to the morphogenetic furrow, and, in addition, there was evidence of reduced neuronal differentiation and abnormal patterning of differentiated cells. Thus, dATM appears to protect proliferating cells from apoptosis and consequently affects their capacity to differentiate (Song, 2004).

In addition to the high level of spontaneous apoptosis observed in dATM mutants, in neuroblasts from dissected brain imaginal discs of dATM mutants, a substantial fraction of mitotic cells exhibit evidence of chromosomal damage, particularly, telomere fusions. Among metaphase chromosomal spreads, both single and double telomere associations were observed, including fusions between sister chromatids as well as between homologous and nonhomologous chromosomes. Occasionally, several chromosomes were fused together to form a chromosome 'chain'. Thus, dATM appears to provide a protective function required for maintenance of telomeres and chromosome structure. Bi (2004) reports that some of the telomere fusions are dependent on ligase IV and are, therefore, likely to occur by a nonhomolgous end-joining mechanism. These observations are of particular interest, because telomere abnormalities have also been observed in mammalian A-T cells. Moreover, it is likely that the presence of dicentric chromosomes, which are unstable, leads to the observed excessive spontaneous apoptosis in dATM mutant tissues during development (Song, 2004).

Like mammalian cells, Drosophila cells respond to DNA damage by undergoing cell cycle arrest. In response to IR, cells of wild-type larval discs exhibit delayed entry into mitosis with increased apoptosis. In mammals, the IR-induced G2 arrest is temporally regulated by the different checkpoint kinases, with ATM playing an important role in the initiation of G2 arrest and ATR playing a more prominent role in maintaining the G2 arrest. Moreover, Chk1 and Chk2 have temporally distinct roles in initiating and maintaining G2 arrest, respectively (Song, 2004 and references therein).

To examine the temporal roles of ATM and mei-41 in Drosophila, mitosis (as an indicator of cell cycle arrest) was examined at various time points after irradiation. Wild-type wing discs had very few mitotic cells 25 min after irradiation, and no mitotic cells were detected at 1 hr postirradiation. The wing disc cells remained arrested and then reentered the cycle approximately 6 hr later. Interestingly, dATM mutant wing discs had significantly more mitotic cells 25 min postirradiation as compared to wild-type discs. At later time points, G2 arrest occurred normally in dATM mutant wing discs. In mei-41 mutant wing discs, cells continued to cycle throughout the time points tested. These results suggest that dATM is involved in the early phase of G2 arrest and mei-41 has a major role in late response. The observed temporal differences in the IR-mediated checkpoint are consistent with those found in mammals (Song, 2004).

Potential functional redundancy between dATM and mei-41 was examined by generating double mutants. Flies harboring mutations in both genes exhibit the same excessive level of spontaneous apoptosis seen in the dATM mutant larval tissues and no apparent increase in apoptosis following IR; however, most of the third-instar larvae exhibited black 'growths', characteristic of so-called melanotic tumors. The underlying basis for these growths is not known, but it suggests that there is some developmental context in which these two genes function redundantly. The mechanisms underlying the distinct and overlapping functions of dATM and mei-41/ATR are likely to involve the upstream activation signals and downstream effector substrates for these closely related kinases; as such, the Drosophila model provides a genetically tractable system that should prove useful in dissecting their function in an in vivo context (Song, 2004).

In conclusion, the function of Drosophila ATM was examined and its role was compared with that of mei-41/ATR during normal development and DNA damage checkpoint responses. Both dATM and mei-41 are highly expressed in female adults and are required for female fertility. Unlike mei-41, flies deficient for dATM exhibit a substantial increase in spontaneous p53-dependent apoptosis and telomere fusions in developing tissues, leading to lethality or to viable flies with malformed adult tissues. It is presumed that dATM deficiency leads to the accumulation of DNA damage during normal cellular replication and differentiation and that this culminates in p53 activation. Although some ATM functions in mammals are mediated by the Chk2 kinase, the essential developmental role of dATM is apparently not mediated by dChk2, which is a nonessential gene, indicating that other ATM substrates are required. In addition to their distinct developmental requirements, dATM and mei-41/ATR perform temporally distinct functions in the DNA damage response to ionizing radiation. Together with the characterization of ATM and ATR functions in mammalian systems, these studies of the Drosophila orthologs point to evolutionarily conserved pathways involving two closely related proteins that together regulate genomic integrity during normal development and in response to genotoxic stress (Song, 2004).

Drosophila atm/telomere fusion is required for telomeric localization of HP1 and telomere position effect

Terminal deletions of Drosophila chromosomes can be stably protected from end-to-end fusion despite the absence of all telomere-associated sequences. The sequence-independent protection of these telomeres suggests that recognition of chromosome ends might contribute to the epigenetic protection of telomeres. In mammals, Ataxia Telangiectasia Mutated (ATM) is activated by DNA damage and acts through an unknown, telomerase-independent mechanism to regulate telomere length and protection. The Drosophila homolog of ATM is encoded by the telomere fusion (tefu) gene. In the absence of ATM, telomere fusions occur even though telomere-specific Het-A sequences are still present. High levels of spontaneous apoptosis are observed in ATM-deficient tissues, indicating that telomere dysfunction induces apoptosis in Drosophila. Suppression of this apoptosis by p53 mutations suggests that loss of ATM activates apoptosis through a DNA damage-response mechanism. Loss of ATM reduces the levels of heterochromatin protein 1 (HP1) at telomeres and suppresses telomere position effect. It is proposed that recognition of chromosome ends by ATM prevents telomere fusion and apoptosis by recruiting chromatin-modifying complexes to telomeres (Oikemus, 2004).

Drosophila atm is required to protect telomeres from fusion. HP1 and HOAP localize to the telomeres of polytene chromosomes, as well as other sites, and are required for telomere protection in mitotic cells. Immunostaining was used to examine the distribution of HP1 and HOAP on wild-type and atm^- polytene chromosomes. HP1 staining at the chromocenter, fourth chromosome, and several euchromatic sites is unaffected by loss of atm, whereas HP1 staining is reduced at most atm^- telomeres. At the tip of chromosome 2R, similar levels of HP1 staining at an internal site (cytological position 60F) can be observed in wild-type and mutant chromosomes, whereas HP1 is specifically reduced at the telomere of the mutant chromosome. The normal levels of HP1 at sites other than the telomere indicate that the lack of telomere staining at atm^- chromosomes is not due to differences in chromosome preparations or to global changes in chromatin structure in atm^- cells. Rather, atm is specifically required to recruit or maintain HP1 to chromosome ends (Oikemus, 2004).

Immunostaining of the same chromosomes for HOAP reveal reduced staining at the telomeres of most atm^- chromosomes compared with wild type. Similar decreases in HP1 and HOAP localization at telomeres are seen in atm^Delta356/ Df(3R)PG4 and atm^tefu/Df(3R)PG4 animals, indicating that this phenotype is not allele specific. Quantification of the fluorescence intensity associated with HOAP and HP1 staining further demonstrates that there is a reproducible reduction at atm^- telomeres compared with wild type. In contrast, HP1 staining at an internal chromosomal site (60F) is not reduced (Oikemus, 2004).

HP1 promotes heterochromatin formation, in part, by recruiting histone-modifying enzymes. To probe whether atm mutations alter chromatin at the telomeres of mitotic cells, telomere position effect (TPE) at three telomeres was examined. When a white reporter gene is placed adjacent to telomere-associated sequences (TAS), gene expression is silenced. At each site tested, TPE is partially suppressed by mutations in atm. In transgenes inserted at nontelomeric genomic positions, placement of the TAS from the telomere of chromosome arm 2L next to the white reporter gene is sufficient to silence white expression. Unlike TAS in their normal location adjacent to telomeres, silencing by a nontelomeric TAS is not affected by atm mutations. These results indicate that the suppression of TPE is due to the specific action of atm on gene expression near telomeres (Oikemus, 2004).

In other organisms, loss of telomere protection can be due to the attrition or degradation of telomere repeat sequences. In Drosophila, it is possible to recover terminal deletions that remove all telomere-specific sequences. However, these observations do not rule out the possibility that telomere-specific sequences contribute to telomere protection or TPE at normal Drosophila telomeres. In fact, the number of telomere repeats has been shown to influence some forms of TPE (Mason, 2003). To test whether the telomere defects in atm^- animals could be due to loss of telomere sequences, fluorescent in situ hybridization was performed using a probe to the Het-A retrotransposon, which is specific to telomere DNA. Hybridization was performed with wild-type and atm^- diploid and polytene chromosomes. In mitotic chromosomes from diploid neuroblast cells, the levels of Het-A hybridization are variable, but not significantly different between wild-type and atm mutant cells. In polytene chromosomes, HeT-A sequences are strongly detected at two telomeres of both wild-type and atm^- chromosomes. Previous analysis of HP1 mutants demonstrated that telomere-specific sequences were still present at chromosome fusion sites (Fanti, 1998). In atm mutant cells, Het-A hybridization is also detected at sites of chromosome fusion. These results indicate that the reduction of telomeric HP1-HOAP and the fusion of telomeres in atm^- cells is not a direct or indirect result of telomere sequence loss (Oikemus, 2004).

Both wild-type and terminally deleted Drosophila chromosomes are protected from telomere fusion and are capped with the telomere-protection proteins HP1 and HOAP (Biessmann, 1988; Fanti, 1998; Cenci, 2003). These results indicate that sequence-independent mechanisms can recruit and maintain telomere protection complexes to chromosome ends. This study has demonstrated that Drosophila atm/tefu is required to prevent chromosome end fusions, to regulate levels of HP1 and HOAP at telomeres, and to promote telomere-position effect. It is also found that atm is required for induction of apoptosis by ionizing radiation. Given the conserved role of ATM family proteins in recognizing DNA breaks, it is suggested that Drosophila ATM protects telomeres by recognizing chromosome ends and recruiting chromatin-modifying proteins to those ends (Oikemus, 2004).

To date ATM protein has not been directly detected at Drosophila telomeres. However, on the basis of results in mammalian cells, it may be necessary to develop antibodies specific for activated forms of ATM to probe ATM activity at telomeres (Bakkenist, 2003). However, several observations presented here indicate that Drosophila ATM acts at telomeres to prevent chromosome fusions. (1) The chromosome rearrangements observed are consistent with a defect in telomere protection rather than translocations due to defective DNA repair or replication. Most chromosome fusions occur near the ends of chromosome arms, and this study demonstrates that the fused chromosomes still contain telomeric DNA sequences. (2) A high frequency of acentric chromosome fragments is not observed during metaphase. In animals mutant for other damage-signaling genes such as the Drosophila homologs of ATR and ATRIP, acentric chromosome fragments are often observed during metaphase, suggesting that these mutations cause a defect in DNA repair or replication that is not observed in atm^- animals. (3) Circular chromosomes do not undergo rearrangements in atm^tefu mutant animals (Queiroz-Machado, 2001), strongly indicating that chromosome fusions are due to fusion of existing chromosome ends rather than the creation of new chromosome breaks. (4) ATM is specifically required for localization of HP1 to telomeres but not centromeric or euchromatic sites. (5) Loss of atm suppresses silencing by telomere-associated sequences when they are adjacent to telomeres, but not when they are at euchromatic sites (Oikemus, 2004).

The telomere fusion defect seen in atm^- animals is consistent with a partial defect in telomere protection. Whereas ~80% of atm^- metaphases contain a chromosome fusion, >95% of metaphases from animals lacking HP1 or HOAP contain a fusion (Fanti, 1998; Cenci, 2003). Furthermore, in some cells lacking HP1 or HOAP, nearly all telomeres appear to be fused. This extreme phenotype has not been observed in atm mutant nuclei. Consistent with a partial defect in telomere protection, the levels of HP1 and HOAP at polytene telomeres are found to be reduced, but not eliminated, in atm^- animals. In mitotic cells, formation of repressive chromatin is also partially disrupted. The interpretation of these results is that reduced and variable levels of HP1 at the telomeres of atm^- animals are sufficient to protect some, but not all telomeres from fusion. The results also indicate that another pathway, possibly involving other DNA damage-response proteins, must contribute to HP1 and HOAP localization, TPE, and telomere protection (Oikemus, 2004).

The direct target of ATM at telomeres is unclear. The decrease in HP1 and HOAP levels at atm^- telomeres is not due to a loss of telomere sequences; wild-type and atm^- chromosomes exhibit similar levels of a telomere-specific retrotransposon sequence as assayed by FISH, and even sites of fusion retain this sequence. This result is consistent with previous demonstrations that the sequences at chromosome ends are not required for telomere protection or for telomeric localization of HP1 and HOAP. Instead, ATM is likely to affect the interaction of HP1 and HOAP with telomeres by regulating the formation of the HP1-HOAP complex or by modification of telomeric chromatin. Other proteins in the DNA damage-response pathway may act with ATM to maintain telomere protection. Although Chk1, Chk2, and p53 are targets of mammalian ATM during the DNA damage response (Shiloh, 2003), Drosophila homologs of these proteins do not appear to be required for telomere protection; animals lacking one or more of these genes do not exhibit the high levels of apoptosis associated with loss of ATM (Brodsky, 2004). Mutations in homologs of other ATM targets such as NBS1 or SMC1 have not been described in Drosophila (Oikemus, 2004).

Recruitment of HP1 and HOAP by ATM is likely to alter chromatin structure at telomeres. HP1 plays a conserved role in heterochromatin formation, histone modification, and gene silencing . In Drosophila, both HP1 and HOAP are required for gene silencing at pericentric heterochromatin. In addition, HP1 is required for gene silencing near fourth chromosome and terminally deleted telomeres, and for repression of P-element transposition by subtelomeric P-element insertions. HP1 homologs are also associated with telomere function in other eukaryotes. In mammals, all three HP1 homologs are found at telomeres, and loss of histone H3 methylases leads to reduced levels of HP1 homologs at telomeres as well as elongated telomeres (Garcia-Cao, 2004). In contrast, overexpression of mammalian HP1 homologs is associated with decreased telomere length (Sharma, 2003). The fission yeast homolog of HP1 is not required for telomere protection, but does regulate telomere length, telomere clustering, and telomeric gene silencing. Interestingly, as in Drosophila telomere protection, some aspects of telomere function in fission yeast are controlled by an epigenetic mechanism. Together, these observations indicate that a requirement for HP1 in telomere function and chromatin structure is conserved, but that its precise role at the telomere may differ among organisms (Oikemus, 2004 and references therein).

Regulation of telomere chromatin structure is also a conserved function of ATM-like kinases. Fission yeast Rad3 and budding yeast Mec1 are required for gene silencing at telomeres (Matsuura, 1999; Craven, 2000) and mutations in human ATM are associated with altered nucleosomal periodicity at telomeres (Smilenov, 1999). The conserved role of ATM-kinases in telomere protection and telomeric chromatin structure suggests that these functions might be linked. The finding that Drosophila ATM is required for TPE and HP1-HOAP localization to telomeres demonstrates one mechanism by which ATM can influence telomere chromatin. It is possible that in organisms that utilize sequence-specific binding proteins such as TRF2 to protect telomeres, regulation of telomeric heterochromatin by ATM and HP1 plays a minor role in protection of normal telomeres, but a more important role at short telomeres that cannot recruit sufficient levels of TRF2. Such a model might explain the synergistic telomere defects seen in cells lacking both telomerase and ATM (Ritchie, 1999; Tsukamoto, 2001; Chan, 2003; Wong, 2003). The lack of an obvious TRF2 homolog may explain why ATM and HP1 play such striking roles in the protection of Drosophila telomeres (Oikemus, 2004).

In addition to preventing chromosome end fusion by DNA repair enzymes, telomere protection is required to prevent activation of DNA damage responses, including the induction of p53-dependent apoptosis and senescence. This analysis of the cellular effects of ATM loss indicates that induction of p53-dependent apoptosis is a conserved consequence of unprotected telomeres in metazoans. Because these unprotected telomeres lead to anaphase bridges and chromosome breaks, p53 may be directly activated by unprotected telomeres or may be activated by subsequent chromosome breaks. Drosophila ATM is required for the induction of apoptosis following IR. Because the spontaneous apoptosis in atm^- animals is, by definition, ATM independent, a different pathway must be able to activate Drosophila p53 in response to unprotected telomeres. Similarly, loss of mammalian ATM reduces, but does not eliminate p53-dependent apoptosis in response to unprotected telomeres (van Steensel, 1998; Takai, 2003; Wong, 2003). Other DNA damage-response pathways may activate Drosophila p53 in the absence of ATM (Oikemus, 2004).

In yeast, insects, and mammals, ATM-kinases are required to activate cellular responses to DNA ends generated by exogenous DNA damage, but also to suppress activation of these pathways by telomeres. Specific recognition of telomere sequences by telomere repeat-binding proteins provides one means to distinguish telomeric DNA ends from damage-induced DNA breaks. However, this mechanism is not sufficient to explain the epigenetic regulation of telomere protection in Drosophila. The requirement of ATM to recruit HP1 and HOAP to Drosophila telomeres suggests that recognition of chromosome ends contributes to chromatin-mediated telomere protection. This model may help explain how terminally deleted chromosomes can be stably inherited without any telomere-specific sequences. Future studies should reveal which other damage response proteins help ATM protect telomeres, what their targets are at telomeres, and how these proteins distinguish between damage-induced DNA ends and the natural ends of chromosomes (Oikemus, 2004).

Drosophila ATM and ATR checkpoint kinases control partially redundant pathways for telomere maintenance

In higher eukaryotes, the ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) checkpoint kinases play distinct, but partially overlapping, roles in DNA damage response. Yet their interrelated function has not been defined for telomere maintenance. The two proteins control partially redundant pathways for telomere protection in Drosophila: the loss of ATM (encoded by telomere fusion) leads to the fusion of some telomeres, whereas the loss of both ATM and ATR (encoded by mei-41) renders all telomeres susceptible to fusion. The ATM-controlled pathway includes the Mre11 and Nijmegen breakage syndrome complex but not the Chk2 kinase, whereas the ATR-regulated pathway includes its partner ATR-interacting protein but not the Chk1 kinase. This finding suggests that ATM and ATR regulate different molecular events at the telomeres compared with the sites of DNA damage. This compensatory relationship between ATM and ATR is remarkably similar to that observed in yeast despite the fact that the biochemistry of telomere elongation is completely different in the two model systems. Evidence is provided suggesting that both the loading of telomere capping proteins and normal telomeric silencing require ATM and ATR in Drosophila and it is proposed that ATM and ATR protect telomere integrity by safeguarding chromatin architecture that favors the loading of telomere-elongating, capping, and silencing proteins (Bi, 2005).

This study defines two partially redundant pathways, regulated by ATM and ATR, respectively, that ensure complete protection of Drosophila telomeres. It is further suggested that two other proteins, Meiotic recombination 11 (Mre11) and Nbs) belong to the same ATM-regulated pathway, whereas ATR-Interacting Protein (ATRIP or Mutagen-sensitive 304) participates in the ATR-controlled pathway. This conclusion would be consistent with the pathway components defined in yeasts. Based on the facts that Drosophila tefu mutants have widespread telomere fusions that eventually lead to lethality, whereas mei-41 mutants are viable with no apparent telomeric defects, it is proposed that ATM is more important in regulating telomere protection, with ATR playing a backup role. In mei-41 mutants, ATM may fully compensate ATR's absence on telomeres to ensure complete capping throughout the cell cycle. In contrast, ATR in tefu mutants may partially compensate for the loss of ATM because it is a less efficient regulator of capping as suggested earlier. This partial redundancy leads to the fusion of some telomeres (Bi, 2005).

ATM, ATR, and their cofactors are known to control multiple checkpoints in response to DNA damage and abnormal telomeres. Perhaps cells with uncapped telomeres are allowed to continue cycling because of defective checkpoints and that leads to the fusion of these telomeres. This possibility is considered unlikely based on several observations. (1) In cav¹ mutant (see caravaggio), telomere fusion occurs at a high rate even in the presence of a full complement of checkpoint genes. (2) No exacerbated telomere dysfunction is found in either the cav¹ tefu (atm) or the cav¹ mei-41 (atr) double mutant. (3) Mutations in Drosophila chk1 or chk2 did not affect existing telomere defects in either tefu or mre11 mutant, suggesting that checkpoints jointly controlled by these effectors and the respective upstream kinases do not normally respond to dysfunctional telomeres. Therefore, the contribution to the telomere dysfunction from checkpoint defects is likely small in the cases studied (Bi, 2005).

Cytological analyses suggest that one of the functions of ATM and ATR at Drosophila telomeres is to facilitate the loading of telomere capping and silencing proteins, such as HOAP (Caravaggio) and HP1, and they do so in a partially redundant fashion. In the case of HOAP's binding to mitotic telomeres, either ATM or ATR is sufficient for its normal loading. When both kinases are absent, HOAP can no longer be detected at the telomeres. It was interesting that there were more telomere fusions in either tefu mei-41^D9 or tefu mus304^D1/D3 than in either mre11 or nbs¹ cells, yet normal HOAP signals were detected on fusion-free telomeres for only the first two genotypes. It is known that the presence of HOAP at chromosome ends is not sufficient to prevent fusion, which suggests that the loss of other capping proteins could also lead to fusion. That may be the case in tefu mei-41^D9 and tefu mus304^D1/D3 cells. It is also known that the absence of HOAP at any particular telomere does not necessarily lead to fusion, suggesting other capping proteins can sometimes compensate for HOAP's function. That idea, in contrast, may apply to the situation in cells deficient for the Mre11 complex (Bi, 2005).

The mechanism by which ATM and ATR maintain telomere integrity remains largely unclear. Because the conserved kinase domain of both ATM and ATR is essential for normal telomere function in S. cerevisiae, ATM and ATR likely exert their function by protein phosphorylation. Neither Chk1 nor Chk2 was involved in telomere protection in Drosophila, a situation similar to S. pombe, which suggests that ATM and ATR modify a different set of proteins at telomeres. ATM and ATR may regulate HOAP's capping activity by directly phosphorylating HOAP. However, no evidence was recovered that HOAP is phosphorylated in WT cells. Therefore, ATM and ATR may indirectly regulate HOAP's ability to bind telomeres, perhaps by modulating telomeric structure. The results support the hypothesis that ATM and ATR have a conserved function at the telomeres that is independent of telomerase. Because Drosophila telomeres are not elongated by a telomerase, the fly may be an excellent system for studying the roles of ATM and ATR in telomere protection, uncoupled from their roles in telomere elongation (Bi, 2005).

The Drosophila Nbs protein functions in multiple pathways for the maintenance of genome stability

Two protein kinases ATM and ATR as well as the Mre11/Rad50/Nbs (MRN) complex, which contains two highly conserved proteins Mre11 and Rad50 and a third less-conserved component, Nbs/Xrs2 (also known as nibrin), play critical roles in the response to DNA damage and telomere maintenance in mammalian systems. The primary function of the MRN complex is to sense DNA strand breaks and then to amplify the initial signal and convey it to downstream effectors, such as ATM, p53, Nbs1 (as a target of ATM), SMC1 and Brca1, that regulate cell cycle checkpoints and DNA repair. Mre11-Rad50 can bind DNA and that Mre11 possesses a nuclease activity that can process these ends. Nbs stimulates the DNA binding and nuclease activity by Mre11-Rad50. In vivo, Nbs is responsible for translocating the MRN complex to the nucleus and relocalizing the complex to the sites of DSBs following irradiation. The MRN complex is also required for activation of the S-phase checkpoint following DNA damage (Ciapponi, 2006).

It has been shown that mutations in the Drosophila mre11 and rad50 genes cause both telomere fusion and chromosome breakage. This study analyzed the role of the Drosophila nbs gene in telomere protection and the maintenance of chromosome integrity. Larval brain cells of nbs mutants display telomeric associations (TAs) but the frequency of these TAs is lower than in either mre11 or rad50 mutants. Consistently, Rad50 accumulates in the nuclei of wild-type cells but not in those of nbs cells, indicating that Nbs mediates transport of the Mre11/Rad50 complex in the nucleus. Moreover, epistasis analysis revealed that rad50 nbs, tefu (ATM) nbs, and mei-41 (ATR) nbs double mutants have significantly higher frequencies of TAs than either of the corresponding single mutants. This suggests that Nbs and the Mre11/Rad50 complex play partially independent roles in telomere protection and that Nbs functions in both ATR- and ATM-controlled telomere protection pathways. In contrast, analysis of chromosome breakage indicated that the three components of the MRN complex function in a single pathway for the repair of the DNA damage leading to chromosome aberrations (Ciapponi, 2006).

This study shows that the wild-type function of the Drosophila nbs gene is required to maintain chromosome integrity and to prevent telomere fusion. The results indicate that the nbs, mre11, and rad50 genes function in single pathway for the repair of spontaneous DNA lesions leading to chromosome breakage. In addition, it was found that double mutants affecting a single component of the MRN complex and either the ATM or the ATR kinase exhibit more chromosome breaks than the corresponding single mutants. The simplest interpretation of these results is that the two kinases function in multiple pathways for the repair of the DNA damage leading to chromosome breakage and that some of these pathways do not include the MRN complex. The finding that mei-41^29D tefu^atm6 double mutants and tefu^atm6 single mutants display similar frequencies of chromosome breaks indicates that the ATM and ATR play redundant roles in the protection from spontaneous chromosome breakage. However, tefu and mei-41 mutants are four- and eightfold more sensitive than wild type to the X-ray induction of chromosome breakage, respectively. Thus, ATR may play a principal role in the repair of the lesions leading to chromosome breaks, with ATM playing a backup role (Ciapponi, 2006).

Recent work has shown that ATM and ATR/ATRIP function in different but redundant pathways of Drosophila telomere protection, with ATM playing an essential role and ATR compensating for the loss of ATM activity. This study shows that the frequencies of TAs observed in nbs tefu and rad50 nbs double mutants are significantly higher than those observed in the corresponding single mutants. An interpretation of these findings is that the Nbs protein functions in a telomere protection pathway that is different from either the ATR/ATRIP or the ATM/Rad50/Mre11 pathway. Alternatively, Nbs could function in both the ATM- and ATR-controlled pathways. These results are at odds with those obtained in budding yeast, where Tel1 (the ATM homolog), Rad50, Mre11, and Xrs2 (the NBS homolog) function in a single pathway of telomere maintenance. However, they are consistent with several results obtained in human cells, showing that the NBS and the MRE11/RAD50 components of the MRN complex can function independently. For example, it has been shown that NBS1 and the MRE11/RAD50 complex have separate roles in both ATM activation and ATM-mediated phosphorylation events. Moreover, while NBS1 localization to the human telomeres is restricted to the S phase, the MRE11/RAD50 complex remains associated with telomeres throughout the cell cycle (Ciapponi, 2006 and references therein).

The results suggest a model for the role of Nbs in Drosophila telomere protection. This model is based on the assumption that Nbs can facilitate both ATR- and ATM-mediated phosphorylation events, as recently shown in mammalian systems. It is proposed that Nbs is involved in both the Rad50/Mre11/ATM and the ATR/ATRIP telomere protection pathways. Nbs would mediate the transport of the Rad50/Mre11 complex in the nucleus in the Rad50/Mre11/ATM pathway and facilitate certain ATR-mediated phosphorylation events in the ATR/ATRIP pathway. Taking into account that the ATR/ATRIP telomere protection pathway is redundant (Bi, 2005), the model can explain the results of the epistasis analysis. It is speculated that in nbs mutants both pathways are partially impaired, resulting in a relatively low frequency of TAs. In rad50 nbs and tefu nbs double mutants, the Rad50/Mre11/ATM pathway would be disrupted and the ATR/ATRIP pathway partially impaired, resulting in TA frequencies higher than those found in the single mutants. Finally, in mei-41 tefu, mus-304 tefu, mei-41 rad50, and mei-41 mre11 double mutants, both pathways would be disrupted, resulting in very high frequencies of TAs (Ciapponi, 2006).

An aspect of the phenotype that is difficult to explain is the pattern of HOAP localization in different mutants and double mutants. In the mre11 and rad50 mutants, most mitotic telomeres are devoid of the HOAP protein. In nbs mutants, the frequency of telomeres with detectable HOAP accumulations is lower than in wild type but higher than in either the mre11 or the rad50 mutant, consistent with a reduced intranuclear concentration of the Rad50/Mre11 complex. tefu (ATM) and mei-41 (ATR) single mutants have normal HOAP concentrations at mitotic telomeres (Bi, 2004) but in mei-41 tefu double mutants telomeres lack the HOAP protein (Bi, 2005). Normal HOAP accumulations at mitotic telomeres were also found in Su(var)205 (HP1) and woc mutants that display very high frequencies of TAs, indicating that the presence of HOAP at chromosome ends is not sufficient to ensure proper telomere protection. An interpretation of these results is rather difficult, mainly because the current knowledge of the Drosophila telomere components is largely incomplete. HOAP localization at telomeres may be mediated, not only by the Rad50/Mre11 complex, but also by a factor that needs to be phosphorylated by both ATM and ATR. When this factor is not phosphorylated at its ATM-dependent site(s), telomeres are deprotected even if they accumulate normal amounts of HOAP. However, when this factor is not phosphorylated in both its ATM- and ATR-dependent sites, telomeres lose their ability to recruit HOAP. This factor cannot be HOAP itself, as recent work (Bi, 2005) has shown that the HOAP protein is not phosphorylated in a wild-type background (Ciapponi, 2006).

This study has shown that the Drosophila Nbs protein is required for transport of Rad50 in the nucleus and for prevention of telomere fusion and chromosome breakage. In addition, the results indicate that Nbs can act independently of the Rad50/Mre11 complex. Remarkably, all these features of the Drosophila Nbs protein are shared by its human counterpart. The ATLD disorder caused by hipomorphic mutations in the MRE11 gene and NBS have many overlapping features but are clinically distinct. NBS patients are characterized by microcephaly and developmental delay, while ATLD patients exhibit a mild ataxia telangiectasia-like phenotype with no microcephaly and no developmental delay. Given the functional similarities within Drosophila and human NBS proteins, it is likely that further studies on the Drosophila MRN complex will help to elucidate the molecular basis of the clinical differences between ATLD and NBS (Ciapponi, 2006 and references therein).

Epigenetic telomere protection by Drosophila DNA damage response pathways: Drosophila nbs is required for both atm- and atr-dependent DNA damage responses and acts in these pathways during DNA repair

Analysis of terminal deletion chromosomes indicates that a sequence-independent mechanism regulates protection of Drosophila telomeres. Mutations in Drosophila DNA damage response genes such as atm/tefu, mre11, or rad50 disrupt telomere protection and localization of the telomere-associated proteins HP1 and HOAP, suggesting that recognition of chromosome ends contributes to telomere protection. However, the partial telomere protection phenotype of these mutations limits the ability to test if they act in the epigenetic telomere protection mechanism. The roles were examined of the Drosophila atm and atr-atrip DNA damage response pathways and the nbs homolog in DNA damage responses and telomere protection. As in other organisms, the atm and atr-atrip pathways act in parallel to promote telomere protection. Cells lacking both pathways exhibit severe defects in telomere protection and fail to localize the protection protein HOAP to telomeres. Drosophila nbs is required for both atm- and atr-dependent DNA damage responses and acts in these pathways during DNA repair. The telomere fusion phenotype of nbs is consistent with defects in each of these activities. Cells defective in both the atm and atr pathways were used to examine if DNA damage response pathways regulate telomere protection without affecting telomere specific sequences. In these cells, chromosome fusion sites retain telomere-specific sequences, demonstrating that loss of these sequences is not responsible for loss of protection. Furthermore, terminally deleted chromosomes also fuse in these cells, directly implicating DNA damage response pathways in the epigenetic protection of telomeres. It is proposed that recognition of chromosome ends and recruitment of HP1 and HOAP by DNA damage response proteins is essential for the epigenetic protection of Drosophila telomeres. Given the conserved roles of DNA damage response proteins in telomere function, related mechanisms may act at the telomeres of other organisms (Oikemus, 2006).

ATM and ATR pathways signal alternative splicing of Drosophila TAF1 pre-mRNA in response to DNA damage

Alternative pre-mRNA splicing is a major mechanism utilized by eukaryotic organisms to expand their protein-coding capacity. To examine the role of cell signaling in regulating alternative splicing, the splicing of the Drosophila TAF1 pre-mRNA was analyzed. TAF1 encodes a subunit of TFIID, which is broadly required for RNA polymerase II transcription. TAF1 alternative splicing generates four mRNAs, TAF1-1, TAF1-2, TAF1-3, and TAF1-4, of which TAF1-2 and TAF1-4 encode proteins that directly bind DNA through AT hooks. TAF1 alternative splicing was regulated in a tissue-specific manner and in response to DNA damage induced by ionizing radiation or camptothecin. Pharmacological inhibitors and RNA interference were used to demonstrate that ionizing-radiation-induced upregulation of TAF1-3 and TAF1-4 splicing in S2 cells is mediated by the ATM (ataxia-telangiectasia mutated) DNA damage response kinase and checkpoint kinase 2 (CHK2), a known ATM substrate. Similarly, camptothecin-induced upregulation of TAF1-3 and TAF1-4 splicing is mediated by ATR (ATM-RAD3 related) and CHK1. These findings suggest that inducible TAF1 alternative splicing is a mechanism to regulate transcription in response to developmental or DNA damage signals and provide the first evidence that the ATM/CHK2 and ATR/CHK1 signaling pathways control gene expression by regulating alternative splicing (Katzenberger, 2006; Full text of article).

Mutations in String/CDC25 inhibit cell cycle re-entry and neurodegeneration in a Drosophila model of Ataxia telangiectasia

Mutations in ATM (Ataxia telangiectasia mutated) result in Ataxia telangiectasia (A-T), a disorder characterized by progressive neurodegeneration. Despite advances in understanding how ATM signals cell cycle arrest, DNA repair, and apoptosis in response to DNA damage, it remains unclear why loss of ATM causes degeneration of post-mitotic neurons and why the neurological phenotype of ATM-null individuals varies in severity. To address these issues, a Drosophila model of A-T was generated. RNAi knockdown of ATM in the eye caused progressive degeneration of adult neurons in the absence of exogenously induced DNA damage. Heterozygous mutations in select genes modified the neurodegeneration phenotype, suggesting that genetic background underlies variable neurodegeneration in A-T. The neuroprotective activity of ATM may be negatively regulated by deacetylation since mutations in a protein deacetylase gene, RPD3, suppressed neurodegeneration, and a human homolog of RPD3, histone deacetylase 2, bound ATM and abrogated ATM activation in cell culture. Moreover, knockdown of ATM in post-mitotic neurons caused cell cycle re-entry, and heterozygous mutations in the cell cycle activator gene String/CDC25 inhibited cell cycle re-entry and neurodegeneration. Thus, it is hypothesized that ATM performs a cell cycle checkpoint function to protect post-mitotic neurons from degeneration and that cell cycle re-entry causes neurodegeneration in A-T (Rimkus, 2008).

The data indicate that ATM knockdown by RNAi causes degeneration of Drosophila post-mitotic photoreceptor neurons. The neurodegeneration phenotype of ATM knockdown flies is similar to that observed in A-T patients. Neurodegeneration in the fly model occurred in the absence of exogenously induced DNA damage, it occurred independently of developmental defects, and it was progressive, increasing in severity as flies aged. Thus, ATM knockdown flies appear to be an appropriate model to study the cellular mechanisms underlying neurodegeneration in A-T (Rimkus, 2008).

A-T is a monogenic disease resulting from mutation of the ATM gene; however, the genetic screen identified second site genes that affect an A-T phenotype, neurodegeneration. Remarkably, independent lines of evidence, from the literature and from the current studies, support the relevance of each of the modifier genes to the mechanism underlying neurodegeneration in A-T (Rimkus, 2008).

ATM is recruited to DSBs by the trimeric MRE11-RAD50-NBS1 (MRN) DNA repair complex, which possesses ATP- dependent nuclease (MRE11) and DNA-tethering (RAD50) activities (Abraham, 2005). Three of the six genes identified in the screen (Stg, RAD50, and PP2A-B') are known components of the ATM signaling pathway that responds to DNA damage in mammals. A role for RAD50 in promoting neurodegeneration is not specific to the eye, since mutation of RAD50 suppress the lethality of Elav-ATMi flies. In addition, mutation of the gene encoding the NBS1 subunit of MRN suppresses the GMR-ATMi rough eye phenotype, suggesting that suppression by RAD50 and NBS1 mutants is due to reduced activity of the MRN complex. Nevertheless, the mechanism underlying suppression of neurodegeneration is unclear since reduced levels of the MRN complex would intuitively be expected to enhance GMR-ATMi phenotypes. One possibility is that the MRN complex is deregulated in the absence of ATM and carries out activities that are lethal to neurons. Finally, PP2A has been shown to dephosphorylate several ATM signaling pathways substrates, including ATM. Mutation of PP2A-B', which encodes a regulatory subunit of the PP2A complex, may enhance neurodegeneration in ATM knockdown flies by affecting the phosphorylation state of ATM substrates (Rimkus, 2008).

Two of the identified genes, MEKK4 and Delta, have potential links to ATM. Mutation of MEKK4 may enhance neurodegeneration in GMR-ATMi flies by allowing cell cycle progression. Published studies suggest a model whereby ATM and MEKK4 pathways collaborate to prevent cell cycle re-entry of post-mitotic neurons by maintaining the latency of CDC25 proteins. Delta encodes a ligand for the Notch receptor, which regulates cell cycle progression and differentiation in many tissues, including the eye. Thus, there may be cross-talk between the ATM and Notch signaling pathways in neurons (Rimkus, 2008).

Finally, studies in cultured cells revealed a direct link between HDAC2, the human homolog of Drosophila RPD3, and ATM. HDAC2 was found to directly associate with ATM and regulate its kinase activity in the absence of exogenously induced DNA damage. Thus, HDAC2 is likely the TSA-sensitive deacetylase that negatively regulates ATM kinase activity. HDAC2 may function by counteracting acetylation of ATM or downstream components of the ATM signaling pathway. It is important to note that although deacetylation of ATM by HDAC2 may regulate ATM activity, HDAC2 is not necessarily an important factor in A-T since the majority of mutations in A-T patients are nonsense or frameshift mutations that result in complete loss or truncation of ATM protein. Nevertheless, the demonstrated physical and functional interactions between HDAC2 and ATM indicate that HDAC2 is an important component of the ATM signaling paradigm and that information garnered from studies of ATM knockdown flies can advance understanding of ATM function in humans (Rimkus, 2008).

Results from the genetic screen predict that A-T patients with mild neurodegeneration will carry heterozygous mutations in suppressor genes, such as CDC25 family members, whereas A-T patients with severe neurodegeneration will carry heterozygous mutations in enhancer genes, such as MEKK4. It will be interesting to see if genes that enhance neurodegeneration in ATM knockdown flies also enhance neurodegeneration in mice. For example, do ATM^-/- MEKK4^+/- mice exhibit progressive degeneration of cerebellar neurons? If so, this would make mice a practical model for studying the neurodegenerative aspects of A-T (Rimkus, 2008).

The data indicate a causal relationship between cell cycle re-entry and neurodegeneration in the Drosophila model of A-T presented in this study. (1) ATM knockdown in photoreceptor neurons resulted in cell cycle re-entry and neurodegeneration, implicating ATM in both processes. (2) Heterozygous mutation of cell cycle regulatory genes Stg/CDC25, Cdk2, dE2F1, and dE2F2 modified the neurodegeneration phenotype of ATM knockdown flies, highlighting the importance of cell cycle regulation in neurodegeneration. (3) Inhibition of cell cycle re-entry by mutation of Stg/CDC25 also inhibited degeneration of ATM knockdown neurons. In contrast, inhibition of neurodegeneration by expression of P35 did not inhibit cell cycle re-entry. (4) Inhibition of neurodegeneration by expression of P35 caused the accumulation of neurons in S/G2/M phases of the cell cycle, indicating that the neurons that re-entered the cell cycle are the ones that degenerated (Rimkus, 2008).

These findings add to a growing literature linking cell cycle re-entry and neurodegeneration. The observations that terminally differentiated neurons are resistant to oncogenic transformation and that brain tumors of neuronal origin rarely occur suggest that cell cycle re-entry of post-mitotic neurons results in death rather than proliferation. In fact, it has been shown in a variety of systems, including flies and humans, that when neurons re-enter the cell cycle, the result is degeneration rather than proliferation and that ectopic cell cycle activation in neurons is sufficient to trigger degeneration. Furthermore, up-regulation of cell cycle genes, such as proliferating cell nuclear antigen, cyclin A, and cyclin B, has been shown to occur in post-mitotic Purkinje and granule cells of A-T patients; and neurons of ATM^-/- mice have been found to undergo DNA replication. Similarly, studies in both fly and mammalian models of Alzheimer’s disease support a causative link between cell cycle re-entry and neurodegeneration. Thus, failure of cell cycle regulation may be a common cause of neurodegenerative disorders, including A-T (Rimkus, 2008).

It is important to keep in mind that equally plausible and nonexclusive models have been put forth for why neurodegeneration occurs in A-T. The oxidative stress model proposes that neurodegeneration occurs as a consequence of increased oxidative stress, and the DNA damage model proposes that neurodegeneration occurs as a consequence of the accumulation of DNA damage. While these models are described as functioning independently, it is unlikely that this is the case. For example, oxidative stress could lead to cell cycle re-entry through several different pathways, and in response to DNA damage, neurons may re-enter the cell cycle before undergoing cell death (Rimkus, 2008).

This paper has described a powerful experimental model in Drosophila to study the molecular mechanisms that underlie neurodegeneration in the human disease A-T. ATM knockdown in flies caused post-mitotic neurons to re-enter the cell cycle and die by programmed cell death. This finding suggests that ATM performs a cell cycle checkpoint function in post-mitotic neurons, as it does in response to DNA damage in proliferating nonneuronal cells and neuroblasts. Heterozygous mutation of Stg/CDC25 suppressed neurodegeneration in ATM knockdown flies and inhibited cell cycle re-entry, suggesting that cell cycle re-entry is causative for neurodegeneration in A-T. In the future, further genetic, cell biological, and molecular analysis of the Drosophila A-T model will allow addressing of unresolved issues, such as the extent to which oxidative stress and DNA damage contribute to neurodegeneration in the absence of ATM, what factors trigger cell cycle re-entry in the absence of ATM, and what factors link cell cycle re-entry to programmed cell death as opposed to cell division in the absence of ATM (Rimkus, 2008).

Loss of the histone variant H2A.Z restores capping to checkpoint-defective telomeres in Drosophila

The conserved histone variant H2A.Z fulfills many functions by being an integral part of the nucleosomes placed at specific regions of the genome. Telomeres cap natural ends of chromosomes to prevent their recognition as double-strand breaks. At yeast telomeres, H2A.Z prevents the spreading of silent chromatin into proximal euchromatin. A role for H2A.Z in capping, however, has not been reported in any organism. This study uncovered such a role for Drosophila H2A.Z. Loss of H2A.Z, through mutations in either its gene or the domino gene for the Swr1 chromatin-remodeling protein, suppressed the fusion of telomeres that lacked the protection of checkpoint proteins: ATM, ATR, and the Mre11-Rad50-NBS complex. Loss of H2A.Z partially restores the loading of the HOAP capping protein, possibly accounting for the partial restoration in capping. It is proposed that, in the absence of H2A.Z, checkpoint-defective telomeres adopt alternative structures, which are permissive for the loading of the capping machinery at Drosophila telomeres (Rong, 2008).

This study shows that loss of H2AvD in Drosophila suppresses fusion of telomeres that lack the protection of conserved checkpoint proteins: ATM, ATR, or MRN. Drosophila H2AvD encodes the functions for both H2A.X and H2A.Z variants that are translated from separate genes in other organisms. By using transgenes that either have or lack H2A.X function, it was established that H2AvD's role in regulating capping resides in its H2A.Z-homologous region. This conclusion is strengthened by the result from analyzing a domino mutation that behaved similarly to an h2AvD mutation. This represents a novel function of H2A.Z that has not been demonstrated in any other organism (Rong, 2008).

It is possible that the effect of h2AvD mutations on fusion frequencies is an indirect effect of transcriptional mis-regulation of genes controlling the repair and/or response to DSBs. This, however, is unlikely since cav mutant cells lacking the HOAP capping component are equally prone to telomere fusion with or without H2A.Z, suggesting that H2AvD mutant telomeres are not refractory to being repaired as DSBs. In addition, an h2AvD mutation was unable to suppress fusion in an atm cav h2AvD triple mutant, suggesting that cav is epistatic to h2AvD. In light of the observation that an h2AvD mutation can partially restore HOAP binding to atm atr double-mutant telomeres, it is suggested that loss of H2AvD might permit more efficient loading of capping proteins and, therefore, more efficient capping (Rong, 2008).

Another hypothesis considered is that H2AvD accumulates at checkpoint-defective telomeres, interfering with the binding of the capping machinery. However, evidence obtained from immuno-localization of H2AvD did not support this hypothesis. H2AvD has an interesting distribution on mitotic chromosomes in wild-type cells in that it is underrepresented in regions commonly considered heterochromatic. Telomeres are generally considered heterochromatic on the basis of their ability to silence nearby genes. However, recent results suggest that the heterochromatic features of Drosophila telomeres reside in the subtelomeric telomere-associated sequence (TAS) repeats and that the retro-transposon arrays at the extreme of chromosome ends possess certain euchromatic features (Biessmann, 2005). This is consistent with the fact that telomeric retro-transposons are actively transcribed to serve as transposition intermediates (Pardue, 2008). Therefore, H2AvD may not be excluded from wild-type telomeric regions, a suggestion supported by a recent genomewide localization study (Mavrich, 2008). Nevertheless, no elevated level of H2AvD was observed at checkpoint-defective telomeres even though these experiments were set up to favor detection of such enrichment. First, the atm atr double mutant - which has the strongest capping defects and on which the h2AvD mutation had the strongest suppressing effect - was included. Second, H2AvD enrichment would have been prominently detected on telomeres from the Y and fourth chromosomes as well as the short arm of the X chromosome on which H2AvD is normally underrepresented. Therefore, it is unlikely that H2AvD interferes with HOAP loading at checkpoint-defective telomeres and that the loss of such interference partly restores capping in h2AvD mutants (Rong, 2008).

Finally, the absence of H2A.Z might allow telomeres to adopt an alternative structure that is permissive to the loading of capping proteins. At S. cerevisiae telomeres, H2A.Z may demarcate the euchromatin-heterochromatin boundary. It may serve a similar function in Drosophila. Interestingly, recent results suggest that the heterochromatic features of Drosophila telomeres might reside in the subtelomeric TAS regions (Biessmann, 2005). It is possible that H2AvD prevents the spreading of TAS-associated heterochromatin into the transposon arrays. In the absence of H2AvD, Drosophila telomeres might adopt a heterochromatin-like structure, which facilitates the loading of capping proteins. This model is purely speculative due to the fact that the structure of Drosophila telomeres is poorly understood. In particular, the structural elements necessary for the loading of capping machinery remain undetermined. Nevertheless, due to the high degree of conservation in H2A.Z variants from different organisms, its role in regulating telomere capping uncovered in this study may also be conserved (Rong, 2008).

Unprotected Drosophila melanogaster telomeres activate the spindle assembly checkpoint

In both yeast and mammals, uncapped telomeres activate the DNA damage response (DDR) and undergo end-to-end fusion. Previous work has shown that the Drosophila HOAP protein, encoded by the caravaggio (cav) gene, is required to prevent telomeric fusions. This study shows that HOAP-depleted telomeres activate both the DDR and the spindle assembly checkpoint (SAC). The cell cycle arrest elicited by the DDR was alleviated by mutations in mei-41 (encoding ATR), mus304 (ATRIP), grp (Chk1) and rad50 but not by mutations in tefu (ATM). The SAC was partially overridden by mutations in zw10 (also known as mit(1)15) and bubR1, and also by mutations in mei-41, mus304, rad50, grp and tefu. As expected from SAC activation, the SAC proteins Zw10, Zwilch, BubR1 and Cenp-meta (Cenp-E) accumulated at the kinetochores of cav mutant cells. Notably, BubR1 also accumulated at cav mutant telomeres in a mei-41-, mus304-, rad50-, grp- and tefu-dependent manner. These results collectively suggest that recruitment of BubR1 by dysfunctional telomeres inhibits Cdc20-APC function, preventing the metaphase-to-anaphase transition (Musarò, 2008).

In most organisms, telomeres contain arrays of tandem G-rich repeats added to the chromosome ends by telomerase. Drosophila telomeres are not maintained by the activity of telomerase, but instead by the transposition of three specialized retrotransposons to the chromosome ends. In addition, whereas yeast and mammalian telomeres contain proteins that recognize telomere-specific sequences, Drosophila telomeres are epigenetically determined, sequence-independent structures. Nonetheless, Drosophila telomeres are protected from fusion events, just as their yeast and mammalian counterparts are. Genetic and molecular analyses have thus far identified eight loci that are required to prevent end-to-end fusion in Drosophila: effete (eff, also known as UbcD1), which encodes a highly conserved E2 enzyme that mediates protein ubiquitination; Su(var)205 and caravaggio (cav), encoding HP1 and HOAP, respectively; the Drosophila homologs of the ATM, RAD50, MRE11A and NBN (also known as NBS1) genes; and without children (woc), whose product is a putative transcription factor (Musarò, 2008).

To determine whether mutations in genes required for telomere capping also affect cell cycle progression, DAPI-stained preparations of larval brains from seven of these eight telomere-fusion mutants were examined. Mutant brains were examined for the mitotic index (MI) and the frequency of anaphases (AF). The mitotic indices observed for the eff, Su(var)205, mre11, rad50, woc and tefu mutants ranged from 0.46 to 0.75, values that were slightly lower than the mitotic index observed for the wild type (0.86). However, brains from cav mutants showed a fourfold reduction of the mitotic index (0.19) with respect to the wild type. cav mutants also had a very low frequency of anaphases (1.7%-1.9%) compared to the wild type (13.2%), whereas in the other mutants, frequency of anaphases ranged from 8.6% to 12.5%. Reductions in both the mitotic index and the frequency of anaphases were rescued by a cav⁺ transgene, indicating that these phenotypes were indeed due to a mutation in cav (Musarò, 2008).

These results prompted a focus on cav mutations in order to determine how unprotected telomeres might influence cell cycle progression. The cav allele used in this study is genetically null for the telomere-fusion phenotype. cav homozygotes and cav¹/Df(3R)crb-F89-4 hemizygotes show very similar mitotic indices and frequencies of anaphases, indicating that cav is also null for these cell cycle parameters. The cav-encoded HOAP protein localizes exclusively to telomeres; cav produces a truncated form of HOAP that fails to accumulate at chromosome ends (Musarò, 2008).

The low frequencies of anaphases observed in cav mutant cells suggest that they may be arrested in metaphase. To confirm a metaphase-to-anaphase block, mitoses were filmed of cav and wild-type neuroblasts expressing the GFP-tagged H2Av histone. Control cells entered anaphase within a few minutes after chromosome alignment in metaphase, whereas cav cells remained arrested in metaphase for the duration of imaging (Musarò, 2008).

It was hypothesized that the cav-induced metaphase arrest was the result of SAC activation. As in all higher eukaryotes, unattached Drosophila kinetochores recruit three SAC protein complexes (Mad1-Mad2, Bub1-BubR1-Cenp-meta and Rod-Zw10-Zwilch) that prevent precocious sister chromatid separation by negatively regulating the ability of Cdc20 to activate the anaphase-promoting complex or cyclosome (APC/C). Mutations in genes encoding components of these complexes lead to SAC inactivation and allow cells to enter anaphase even if the checkpoint is not satisfied. To ask whether the low frequency of anaphases in cav mutant brains was due to SAC activation, zw10 cav and bubR1 cav double mutants were analyzed. In both cases, the frequency of anaphases was significantly higher than in the cav single mutant, whereas the frequency of telomere fusions remained unchanged. These results imply that the low frequency of anaphases in cav mutants is indeed due to SAC activation (Musarò, 2008).

SAC activation would be expected to increase the mitotic index through the accumulation of metaphase cells; however, in cav single mutants, the mitotic index is abnormally low. One explanation for this apparent paradox is that the cell cycle in cav cells is also delayed before M-phase, as a result of the DNA damage response (DDR). To ask whether HOAP-depleted telomeres activate any DNA damage checkpoints, double mutants were generated for cav and genes known to be involved in these checkpoints: mei-41 and telomere fusion (tefu), encoding the fly homologs of ATR and ATM, respectively; mus304, which encodes the ATR-interacting protein ATRIP grapes (grp), which specifies a CHK1 homolog and rad50, whose product is part of the Mre11-Rad50-Nbs complex. DAPI-stained preparations of larval brain cells from these double mutants showed that mei-41, mus304, grp and rad50 mutations alleviate the cell cycle block induced by cav, causing a ~2.5-fold increase of the mitotic index relative to that observed in the cav single mutant. In contrast, the tefu mutation did not affect the cav- induced interphase block. These effects are unrelated to variations in the frequency of telomere fusions, as the telomere fusion frequencies in double mutants were very similar to those in the cav single mutant. It is thus concluded that the interphase arrest in cav mutants occurs independently of ATM and is mediated by a signaling pathway involving ATR, ATRIP, Chk1 and Rad50. This signaling pathway is known to activate DNA damage checkpoints during the G1/S transition, the S phase and the G2/M transition. However, the current results do not allow identification of the particular checkpoint(s) activated by HOAP-depleted telomeres (Musarò, 2008).

Notably, in all double mutants for cav and any one of the genes associated with the DDR, including tefu (ATM), a significant increase was also observed in the frequencies of anaphases relative to that of the cav single mutant, suggesting that these genes are involved in the cav-induced metaphase arrest. This finding reflects a role of these DDR-associated genes in the peculiar mechanism by which uncapped Drosophila telomeres activate SAC (Musarò, 2008).

To obtain further insight about the cav-induced metaphase arrest, the localization of Zw10, Zwilch, BubR1 and Cenp-meta (Cenp-E) was determined by immunofluorescence. In wild-type Drosophila cells, these proteins begin to accumulate at kinetochores during late prophase and remain associated with kinetochores until the chromosomes are stably aligned at the metaphase plate. Treatments with spindle poisons (for example, colchicine) disrupt microtubule attachment to the kinetochores, leading to metaphase arrest with SAC proteins accumulated at the centromeres. Immunostaining for Zwilch, Zw10, Cenp-meta or BubR1 showed that in all cases, the frequencies of cav metaphases with strong centromeric signals were comparable to those observed in colchicine-treated wild-type cells, and they were significantly higher than those seen in untreated wild-type metaphases. These findings support the view that HOAP-depleted telomeres activate the canonical SAC pathway (Musarò, 2008).

Through a detailed examination of cav metaphases immunostained for SAC proteins, an unexpected connection was found between uncapped telomeres and the localization of at least one SAC component. Although Zwilch, Zw10 and Cenp-meta accumulated exclusively at kinetochores, BubR1 was concentrated at both kinetochores and telomeres. BubR1 localized at both unfused (free) and fused telomeres; most (94.4%) cav metaphases showed at least one telomeric BubR1 signal. To better resolve the chromosome tangles seen in cav metaphases, cells were treated with hypotonic solution, allowing a focus on free telomeres, which can be reliably scored. It was found that 25% of the free telomeres in cav metaphases show an unambiguous BubR1 signal. BubR1 accumulations were not observed at wild-type telomeres or at the breakpoints of X-ray-induced chromosome breaks. BubR1 localization at telomeres was not caused by the formation of ectopic kinetochores at the chromosome ends, since cav telomeres did not recruit the centromere and kinetochore marker Cenp-C. Low frequencies of BubR1-labeled telomeres were also observed in other mutant strains with telomere fusions including eff, Su(var)205 and woc. These results indicate that BubR1 specifically localizes at uncapped telomeres (Musarò, 2008).

It was next asked whether mutations in mei-41, grp, mus304, tefu, rad50 and zw10 affect BubR1 localization at cav mutant telomeres. Whereas mutations in zw10 did not affect BubR1 localization at cav chromosome ends, double mutants for cav and any of the other genes all showed significant reductions in the frequency of BubR1-labeled free telomeres with respect to cav single mutants. Considered together, these results indicate that when the canonical SAC machinery is intact (in all cases except in zw10 cav double mutants), there is a strong negative correlation between the frequency of BubR1-labeled telomeres and the frequency of anaphases. These findings suggest that BubR1 accumulation at telomeres can activate the SAC (Musarò, 2008).

Finally it was asked whether mutations in DDR-associated genes can allow cells to bypass the SAC when it is activated by spindle abnormalities rather than by uncapped telomeres. The spindle was disrupted in two ways: with the microtubule poison colchicine and with mutations in abnormal spindle (asp). Both situations activated the SAC and caused metaphase arrest; neither mei-41 nor grp or tefu mutations allowed cells to bypass this arrest, whereas mutations in zw10 led such cells to exit mitosis. These findings indicate that the DDR-associated genes regulate BubR1 accumulation at cav telomeres but are not directly involved in the SAC machinery (Musarò, 2008).

Collectively, these results suggest a model for the activation of cell cycle checkpoints by unprotected Drosophila telomeres. It is proposed that uncapped telomeres activate DDR checkpoints, leading to interphase arrest through a signaling pathway involving mei-41 (encoding ATR), mus304 (ATRIP), grp (Chk1) and rad50, but not tefu (ATM). This pathway is independent of telomeric BubR1, because mutations in tefu, which strongly reduce BubR1 accumulation at chromosome ends, do not rescue cav-induced interphase arrest. Uncapped telomeres can also activate the SAC by recruiting BubR1 through a pathway requiring mei-41, mus304, grp, rad50 and tefu functions. Once accumulated at the telomeres, BubR1 may negatively regulate either Fizzy (Cdc20) or another APC/C subunit so as to cause metaphase arrest. This model posits that certain DDR-associated genes, such as rad50, function both in the DDR pathway and in the pathway that mediates BubR1 recruitment at telomeres. This explains why rad50 and mre11 mutants show only mild reductions of the mitotic index and the frequency of anaphases even though HOAP is substantially depleted from their telomeres (Musarò, 2008).

It is proposed that uncapped telomeres can induce an interphase arrest independently of BubR1 through a signaling pathway that involves ATR, ATRIP, CHK1 and Rad50 but not ATM. The same proteins, including ATM, are required for the recruitment of BubR1 at unprotected telomeres. Telomeric BubR1 may negatively regulate the activity of the Cdc20-APC complex, leading to a metaphase-to-anaphase transition block. The metaphase arrest caused by Cdc20-APC inhibition is likely to cause an accumulation of SAC proteins on the kinetochores, reinforcing SAC activity. Consistent with this view, mutations in ida, which encodes an APC/C subunit, lead to a metaphase arrest phenotype with BubR1 accumulated at the kinetochores (Musarò, 2008).

Several recent reports have suggested possible relationships between DNA damage, SAC and telomeres. In both Drosophila and mammalian cells, DNA breaks can activate the SAC, presumably by disrupting kinetochore function. In Schizosaccharomyces pombe, Taz1-depleted telomeres result in Mph1p- and Bub1p-mediated SAC activation, and mutations in yKu70 affecting Saccharomyces cerevisiae telomere structure also activate the SAC. However, these previous studies did not explain how telomere perturbations might be perceived by the SAC. This study has found that unprotected Drosophila telomeres recruit the BubR1 kinase as do the kinetochores that are unconnected to spindle microtubules. Thus, it is possible that telomere-associated BubR1 inhibits anaphase through molecular mechanisms similar to those that govern SAC function at the kinetochore. Consistent with this possibility, a single BubR1 accumulation at either a centromere or a telomere seems competent to block anaphase onset. It will be of interest in the future to establish whether deprotected mammalian telomeres can also activate the SAC and, if so, whether BubR1 recruitment to the damaged telomeres mediates this response (Musarò, 2008).

Drosophila ATM and ATR have distinct activities in the regulation of meiotic DNA damage and repair

Ataxia telangiectasia-mutated (ATM) and ataxia telangiectasia-related (ATR) kinases are conserved regulators of cellular responses to double strand breaks (DSBs). During meiosis, however, the functions of these kinases in DSB repair and the deoxyribonucleic acid (DNA) damage checkpoint are unclear. This paper shows that ATM and ATR have unique roles in the repair of meiotic DSBs in Drosophila. ATR mutant analysis indicated that it is required for checkpoint activity, whereas ATM may not be. Both kinases phosphorylate H2AV (γ-H2AV), and, using this as a reporter for ATM/ATR activity, it was found that the DSB repair response is surprisingly dynamic at the site of DNA damage. γ-H2AV is continuously exchanged, requiring new phosphorylation at the break site until repair is completed. However, most surprising is that the number of γ-H2AV foci is dramatically increased in the absence of ATM, but not ATR, suggesting that the number of DSBs is increased. Thus, it is concluded that ATM is primarily required for the meiotic DSB repair response, which includes functions in DNA damage repair and negative feedback control over the level of programmed DSBs during meiosis (Joyce, 2011).

ATR-dependent checkpoint activity in response to unrepaired DSBs causes oocyte development to proceed abnormally. A previous study noted that tefu mutants produced embryos with dorsal-ventral polarity defects, a possible indicator of elevated DSB repair checkpoint activity. Another reporter for this effect is Gurken (GRK), a TGF-α-related protein required for establishing dorsal-ventral polarity. When DSBs are not repaired, GRK localization is abnormal (Joyce, 2011).

At the restrictive temperature (25°C), tefu⁸ mutants are recessive lethal. To examine whether the meiotic DSB repair checkpoint was active in tefu⁸ mutants, homozygous females were raised at the permissive temperature (18°), shifted to the restrictive temperature, and whether there was a disruption of GRK localization was examined. GRK is normally concentrated in the cytoplasm of control oocytes. In 87% of similarly staged tefu⁸ mutant ovarioles, GRK expression was absent or much weaker than normal and mislocalized. Another characteristic feature of oocyte development is the assembly of the karyosome, in which the chromatin is condensed into a single round mass within the cell nucleus of stage 4 oocytes. In control oocytes, the karyosome appeared compact and spherical. However, in 80% of the tefu⁸ mutant oocytes, the karyosome appeared abnormally flattened or fragmented. Abnormal GRK localization and karyosome organization are ATR-dependent phenotypes that are typical of mutants unable to repair DSBs. ATM is required for the completion of meiotic recombination but is dispensable for the DSB repair checkpoint (Joyce, 2011).

MEI-W68 is the Drosophila homologue of Spo11, a conserved endonuclease that catalyzes meiotic DSB induction in eukaryotes. The GRK localization and karyosome morphology defects were suppressed in mei-W86⁴⁵⁷²;tefu⁸ double mutants, indicating that the defects are a result of unrepaired meiotic DSBs. A double mutant genotype combination was tested with mei-41, the Drosophila homologue of ATR. The GRK mislocalization and karyosome defects in tefu⁸ mutants were suppressed in mei-41^D3;tefu⁸ double mutants. These results show that loss of ATM function leads to activation of the ATR-dependent checkpoint response to unrepaired meiotic DSBs (Joyce, 2011).

Drosophila H2A variant, like mammalian H2AX, that is phosphorylated at the sites of DNA breaks. Antibodies to this phosphorylated protein (γ-H2AV) detect distinctive foci in the nucleus. To assay for DSB repair defects in tefu⁸ mutants, γ-H2AV staining was examined and compared with wild-type and mutants known to have DSB repair defects. Pachytene oocytes are arranged in order of developmental age within the germarium, which is divided into three regions. In wild-type females, a mean of 6.2 γ-H2AV foci was found in region 2a pachytene oocytes and was absent in region 3 oocytes. This is consistent with previous results suggesting that meiotic DSBs in wild-type oocytes are induced in region 2a and repaired before region 3 (Joyce, 2011).

Mutations in DSB repair genes such as spn-A (which encodes the Drosophila Rad51 homologue) exhibit an accumulation of γ-H2AV foci that persist throughout meiotic prophase, corresponding to unrepaired meiotic DSBs. A mean of 22.8 γ-H2AV foci was present in spn-A¹ region 3 oocytes, which is similar to previous estimates for the total number of DSBs per nucleus. Similarly, γ-H2AV foci accumulated in region 3 oocytes of mei-41^D3 mutants, indicating that ATR is required to repair meiotic DSBs in addition to its role in checkpoint activation. In tefu⁸ mutant germaria at the restrictive temperature, γ-H2AV staining persisted into region 3 oocytes, consistent with a DSB repair defect. However, in contrast to other repair mutants and wild type, the γ-H2AV staining in tefu⁸ mutants exhibited more robust and continuous labeling, colocalizing with most of the chromosomes rather than appearing as foci. All γ-H2AV staining was eliminated in mei-W86⁴⁵⁷²;tefu⁸ double mutants, indicating that the abundant γ-H2AV staining in the tefu⁸ mutant is dependent on the induction of meiotic DSBs (Joyce, 2011).

The threadlike γ-H2AV labeling observed in tefu⁸ mutant oocytes could be a result of either unrestricted spreading of H2AV phosphorylation from the DSB sites or an increase in the number of programmed DSBs relative to wild type. These possibilities were investigated by examining the nurse cells in the germarium. Each pro-oocyte has 14 neighboring nurse cells that experience on average twofold less DSBs than the oocyte. At the restrictive temperature, tefu⁸ mutants exhibited distinct γ-H2AV foci in nurse cells, indicating that ATM-deficient cells can restrict their DSB response to the DSB sites, and the foci could be counted. The tefu⁸ mutant nurse cells had a mean of 9.3 γ-H2AV foci, which is >2.5 times greater than the 3.6 γ-H2AV foci per nurse cell nurse in wild typ. To estimate the total number of DSBs that occur in tefu⁸ mutant oocytes, a method was used that quantitatively measures the intensity of γ-H2AV fluorescence. In short, the intensity of a single γ-H2AV focus in adjacent nurse cells was compared with that of total fluorescence in oocytes. Based on this method, 25.2 γ-H2AV foci was found in spn-A region 3 oocytes, similar to the levels when counted manually. In tefu⁸ mutants, ~39.1 γ-H2AV foci (P = 0.0152) was estimated, a significant increase over spn-A that is consistent with the increase in γ-H2AV foci levels observed in nurse cells. Together, these results reveal a novel role for ATM in negatively regulating DSB formation during meiotic prophase (Joyce, 2011).

ATM and ATR have been implicated in the phosphorylation of H2AX at sites of chromosomal DSBs in somatic cells of mouse and humans. To investigate whether Drosophila ATM and ATR serve redundant roles in H2AV phosphorylation in response to meiotic DSBs, mei-41^D3;tefu⁸ double mutant germaria were examined. At a permissive temperature (18°), mei-41^D3;tefu⁸ displayed a γ-H2AV staining pattern similar in severity to mei-41^D3 single mutants with a mean of 18.2 foci in region 3 oocytes. When shifted to the restrictive temperature (25°) for 24 h, no γ-H2AV staining was observed in the mei-41^D3;tefu⁸ region 2a cysts, indicating that these mutants lost the ability to phosphorylate H2AV near newly generated DSBs. This is the first demonstration that ATM and ATR are redundant for the phosphorylation of H2AV in response to meiotic DSBs and is consistent with a study in somatic cells of other organisms (Joyce, 2011).

The absence of γ-H2AV staining from mei-41^D3;tefu⁸ double mutant region 2a oocytes indicated that there was no phosphorylation in response to a DSB. However, γ-H2AV was also absent from older region 3 oocytes, indicating that γ-H2AV was lost from DSB sites after only 24 h at the restrictive temperature. That is, based on previous estimates for the timing of cyst progression (12-24 h per region), the region 3 oocytes were in region 2b (after DSB formation) at permissive temperature and would have had γ-H2AV staining before the shift to restrictive temperature. The loss of γ-H2AV staining upon shift to restrictive temperature indicates that there is a rapid turnover of the phosphorylation mark near meiotic DSBs. To confirm that the histone H2AV and DSBs were still present in region 3 nuclei, the mei-41^D3;tefu⁸ double mutants were transferred from the restrictive temperature back to the permissive temperature and γ-H2AV staining was analyzed. After only 24 h at the permissive temperature, γ-H2AV staining returned to the double mutant oocytes, consistent with the presence of unrepaired DSBs and H2AV in region 3 oocytes. These findings indicate that γ-H2AV at meiotic DSB sites is continuously exchanged or dephosphorylated independent of repair and that rephosphorylation of H2AV is maintained by continuous ATM or ATR activity (Joyce, 2011).

The aforementioned results suggest that a component of the DSB repair response involves dynamic changes in chromatin structure, which may be important to maintain ATM/ATR activity until the DSB is repaired. To investigate the mechanism behind the repair-independent constitutive exchange of γ-H2AV, factors known to regulate H2AV exchange in other cell types were examined. In particular, the exchange of γ-H2AV with unphosphorylated H2AV in somatic cells is preceded by the acetylation of the histone by the multiprotein complex (see Tip60). Whether the Tip60 complex component MRG15 is required for γ-H2AV exchange was determined by creating MRG15 mutant germline clones and analyzing H2AV levels throughout oogenesis. Strikingly, a complete absence of H2AV, both phosphorylated and unphosphorylated, was observed in MRG15^j6A3 mutant cells throughout oogenesis. Mutant germline clones are generated in the premeiotic stem cells; therefore, these results indicate that MRG15 is required for the incorporation of H2AV into meiotic chromatin. With this function, MRG15 could also be required for a process that promotes γ-H2AV turnover during meiotic prophase by incorporating unphosphorylated H2AV into the nucleosomes after γ-H2AV has been removed (Joyce, 2011).

>In addition to the acetyltransferase Tip60, MRG15 has been found in another complex that includes the deacetylase Rpd3. Germline clones were made of Rpd3⁰⁴⁵⁵⁶, and it was found that, rather than loss of H2AV, there was abundant γ-H2AV foci and evidence of a repair defect. These results suggest that the Rpd3 complex is not required for H2AV exchange in the germline. Although the Tip60 complex is a strong candidate for this role, confirmation awaits the analysis of additional Tip60 complex components or the construction of Tip60 mutants (Joyce, 2011).

This evidence indicates that γ-H2AV is surprisingly dynamic, being constantly exchanged in a DSB-independent manner. A previous observation was confirmed and extended that in mutants with a defect in DSB repair, such as spn-A¹, mei-41^D3, and tefu⁸, γ-H2AV labeling persists until stage 5 and yet is never observed in more advanced stages of oogenesis. It was reasoned that this absence of γ-H2AV staining past stage 5 may reflect either a reduction in ATM/ATR activity, use of an alternative repair pathway, or loss of the H2AV substrate from the nucleosomes (Joyce, 2011).

To evaluate the presence of histone H2AV in nucleosomes during oogenesis, ovaries were stained with an H2AV antibody that recognizes both phosphorylated and unphosphorylated versions of the histone variant. As expected, H2AV labeling was abundant throughout the nucleus of all oocytes and nurse cells as well as mitotically dividing follicle cells from the germarium to stage 3 of oogenesis. Strikingly, at stage 4-5 of oogenesis, H2AV staining was drastically reduced in nurse cells and oocytes but not in follicle cells. This correlates well with the disappearance of γ-H2AV foci in both the oocyte and nurse cells at this stage in repair mutants. Indeed, the absence of H2AV at stage 5 was also found in spn-A¹, mei-41^D3, and tefu⁸ mutant ovarioles. Therefore, the loss of γ-H2AV signal at stage 5 of oogenesis is a result of the removal of H2AV. Similar results were observed with an H2AV:GFP fusion protein in oocytes, although the signal persisted longer in the nurse cells. These results have important implications for using γ-H2AV as a DSB reporter late in prophase, as it is impossible to determine whether ATM/ATR responds to DNA damage or whether that damage is repaired before the first meiotic division (Joyce, 2011).

This study has shown that the Drosophila ATM and ATR kinases have distinct roles in meiotic DSB repair, results that are consistent with the role of ATM in the mouse germline. Unlike ATR, however, ATM is dispensable for the meiotic DSB repair checkpoint, although it cannot be ruled out a minor role for ATM in the checkpoint because mei-41 mutants fail to completely suppress the effects of some DSB repair mutants. Interestingly, in Drosophila somatic cells, ATM is required for a checkpoint response only at low doses of radiation. Thus, the amount of damage may be high enough in meiotic cells such that ATR signaling is sufficient for the checkpoint response. An alternative is that the number of breaks is not as significant as how they are processed. DSBs experience rapid resection in meiosis to generate single-stranded DNA, which is necessary for ATR activation (Joyce, 2011).

ATM and ATR kinases clearly have common targets, such as the phosphorylation of H2AV. Using γ-H2AV as a reporter, a surprising dynamic component to this phosphorylation was found including at least two phases of H2AV clearance in the Drosophila female germline. First, γ-H2AV at meiotic DSB sites is rapidly exchanged with unphosphorylated H2AV. Because γ-H2AV is exchanged with H2AV independent of DSB repair, the removal of γ-H2AV from DSB sites after repair may only require the cessation of ATM and ATR activity. Second, most of the H2AV is removed between stages 5 and 6 of oogenesis (after pachytene) and occurs independently of the repair and phosphorylation state (Joyce, 2011).

The most surprising result of this study is that ATM negatively regulates meiotic DSB formation. Induction of DSBs is essential to generate crossovers. Approximately 20 DSBs occur per meiosis in Drosophila, but only six or seven become crossovers. Similarly, in yeast and mice, a surplus of DSBs is generated to produce crossovers. What remains unknown are the mechanisms that limit the number of DSBs to prevent excessive genomic damage. It is suggested that ATM is part of a negative feedback mechanism to limit the total number of DSBs. This mechanism of DSB regulation appears to be conserved, as DSB levels are also increased in mouse spermatocytes lacking ATM, which may explain circumstances in which crossovers are increased in the absence of ATM (Joyce, 2011).

Contributions of DNA repair, cell cycle checkpoints and cell death to suppressing the DNA damage-induced tumorigenic behavior of Drosophila epithelial cells

When exposed to DNA-damaging agents, components of the DNA damage response (DDR) pathway trigger apoptosis, cell cycle arrest and DNA repair. Although failures in this pathway are associated with cancer development, the tumor suppressor roles of cell cycle arrest and apoptosis have recently been questioned in mouse models. Using Drosophila epithelial cells that are unable to activate the apoptotic program, evidence is provided that ionizing radiation (IR)-induced DNA damage elicits a tumorigenic behavior in terms of E-cadherin delocalization, cell delamination, basement membrane degradation and neoplasic overgrowth. The tumorigenic response of the tissue to IR is enhanced by depletion of Okra/DmRAD54 or spnA/DmRAD51-genes required for homologous recombination (HR) repair of DNA double-strand breaks in G2-and it is independent of the activity of Lig4, a ligase required for nonhomologous end-joining repair in G1. Remarkably, depletion of Grapes/DmChk1 or Mei-41/dATR-genes affecting DNA damage-induces cell cycle arrest in G2-compromises DNA repair and enhances the tumorigenic response of the tissue to IR. On the contrary, DDR-independent lengthening of G2 has a positive impact on the dynamics of DNA repair and suppressed the tumorigenic response of the tissue to IR. These results support a tumor suppressor roles of apoptosis, DNA repair by HR and cell cycle arrest in G2 in simple epithelia subject to IR-induced DNA damage (Dekanty, 2014).

EVOLUTIONARY HOMOLOGS

ATM in plants

The function of ATM and ATR at telomeres has been examined in Arabidopsis. Although plants lacking ATM or ATR display wild-type telomere length homeostasis, chromosome end protection is compromised in atm atr mutants. Moreover, atm tert Arabidopsis (TERT is the catalytic subunit of telomerase) experience an abrupt, early onset of genome instability, arguing that ATM is required for protection of short telomeres. The rate of telomere shortening is indistinguishable between atm tert and tert mutants, with telomeres declining by ~500 bp per plant generation in both settings. ATR, by contrast, is required for maintenance of telomeric DNA; telomere shortening is dramatically accelerated in atr tert mutants relative to tert plants. Thus, ATM and ATR make essential and distinct contributions to chromosome end protection and telomere maintenance in higher eukaryotes (Vespa, 2005).

Targets of ATM in yeast

Mutants of the Saccharomyces cerevisiae ataxia telangiectasia mutated (ATM) homolog MEC1/SAD3/ESR1 were identified that could live only if the RAD53/SAD1 checkpoint kinase was overproduced. MEC1 and a structurally related gene, TEL1, have overlapping functions in response to DNA damage and replication blocks that in mutants can be provided by overproduction of RAD53. Both MEC1 and TEL1 were found to control phosphorylation of Rad53p in response to DNA damage. These results indicate that RAD53 is a signal transducer in the DNA damage and replication checkpoint pathways and functions downstream of two members of the ATM lipid kinase family. Because several members of this pathway are conserved among eukaryotes, it is likely that a RAD53-related kinase will function downstream of the human ATM gene product and play an important role in the mammalian response to DNA damage (Sanchez, 1996).

DNA damage checkpoint pathways sense DNA lesions and transduce the signals into appropriate biological responses, including cell cycle arrest, induction of transcriptional programs, and modification or activation of repair factors. The Saccharomyces cerevisiae Sae2 protein, known to be involved in processing meiotic and mitotic double-strand breaks, is required for proper recovery from checkpoint-mediated cell cycle arrest after DNA damage and is phosphorylated periodically during the unperturbed cell cycle and in response to DNA damage. Both cell cycle- and DNA damage-dependent Sae2 (SUMO-1-activating enzyme 2) phosphorylation requires the main checkpoint kinase, Mec1, and the upstream components of its pathway, Ddc1, Rad17, Rad24, and Mec3. Another pathway, involving Tel1 and the MRX complex, is also required for full DNA damage-induced Sae2 phosphorylation, that is instead independent of the downstream checkpoint transducers Rad53 and Chk1, as well as of their mediators Rad9 and Mrc1. Mutations altering all the favored ATM/ATR phosphorylation sites of Sae2 not only abolish its in vivo phosphorylation after DNA damage but also cause hypersensitivity to methyl methanesulfonate treatment, synthetic lethality with RAD27 deletion, and decreased rates of mitotic recombination between inverted Alu repeats, suggesting that checkpoint-mediated phosphorylation of Sae2 is important to support its repair and recombination functions (Baroni, 2004).

ATM and telomeres in yeast

The Schizosaccharomyces pombe checkpoint gene named rad3⁺ encodes an ATM-homologous protein kinase that shares a highly conserved motif with proteins involved in DNA metabolism. Previous studies have shown that Rad3 fulfills its function via the regulation of the Chk1 and Cds1 protein kinases. A novel role is described for Rad3 in the control of telomere integrity. Mutations in the rad3⁺ gene alleviate telomeric silencing and produce shortened lengths in the telomere repeat tracts. Genetic analysis has revealed that the other checkpoint rad mutations (rad1, rad17, and rad26) belong to the same phenotypic class with rad3 with regard to control of the telomere length. Of these mutations, rad3 and rad26 have a drastic effect on telomere shortening. tel1⁺, another ATM homolog in S. pombe, carries out its telomere maintenance function in parallel with the checkpoint rad genes. Furthermore, either a single or double disruption of cds1(+) and chk1⁺ causes no obvious changes in the telomeric DNA structure. These results demonstrate a novel role of the S. pombe ATM homologs that is independent of chk1⁺ and cds1⁺ (Matsuura, 1999).

Yeast strains with a mutation in the MEC1 gene (an ATM homolog) are deficient in the cellular checkpoint response to DNA-damaging agents and have short telomeres. In wild-type yeast cells, genes inserted near the telomeres are transcriptionally silenced. mec1 strains have reduced ability to silence gene expression near the telomere. This deficiency was alleviated by the sml1 mutation. Overexpression of Mec1p also results in a silencing defect, although this overexpression does not affect the checkpoint function of Mec1p. Telomeric silencing is not affected by mutations in several other genes in the Mec1p checkpoint pathway (null mutations in RAD9 and CHK1 or in several hypomorphic rad53 alleles) but is reduced by a null mutation of DUN1. In addition, the loss of telomeric silencing in mec1 strains was not a consequence of the slightly shortened telomeres observed in these strains (Craven, 2000).

The Saccharomyces Mre11p, Rad50p, and Xrs2p proteins form a complex, called the MRX complex, that is required to maintain telomere length. Cells lacking any one of the three MRX proteins and Mec1p, an ATM-like protein kinase, undergo telomere shortening and ultimately die, phenotypes characteristic of cells lacking telomerase. The other ATM-like yeast kinase, Tel1p, appears to act in the same pathway as MRX: mec1 tel1 cells have telomere phenotypes similar to those of telomerase-deficient cells, whereas the phenotypes of tel1 cells are not exacerbated by the loss of a MRX protein. The nuclease activity of Mre11p was found to be dispensable for the telomerase-promoting activity of the MRX complex. The association of the single-stranded TG1-3 DNA binding protein Cdc13p with yeast telomeres occurs efficiently in the absence of Tel1p, Mre11p, Rad50p, or Xrs2p. Targeting of catalytically active telomerase to the telomere suppresses the senescence phenotype of mec1 mrx or mec1 tel1 cells. Moreover, when telomerase is targeted to telomeres, telomere lengthening is robust in mec1 mrx and mec1 tel1 cells. These data rule out models in which the MRX complex is necessary for Cdc13p binding to telomeres or in which the MRX complex is necessary for the catalytic activity of telomerase. Rather, the data suggest that the MRX complex is involved in recruiting telomerase activity to yeast telomeres (Tsukamoto, 2001).

Telomerase is a ribonucleoprotein that copies a short RNA template into telomeric DNA, maintaining eukaryotic chromosome ends and preventing replicative senescence. Telomeres differentiate chromosome ends from DNA double-stranded breaks. Nevertheless, the DNA damage-responsive ATM kinases Tel1p and Mec1p are required for normal telomere maintenance in Saccharomyces cerevisiae. Tests were performed to see whether the ATM kinases are required for telomerase enzyme activity or whether it is their action on the telomere that allows telomeric DNA synthesis. Cells lacking Tel1p and Mec1p had wild-type levels of telomerase activity in vitro. Furthermore, altering telomere structure in three different ways showed that telomerase can function in ATM kinase-deleted cells: tel1 mec1 cells senesced more slowly than tel1 mec1 cells that also lacked TLC1, which encodes telomerase RNA, suggesting that tel1 mec1 cells have residual telomerase function; deleting the telomere-associated proteins Rif1p and Rif2p in tel1 mec1 cells prevented senescence; a point mutation was isolated in the telomerase RNA template domain (tlc1-476A) that altered telomeric DNA sequences, causing uncontrolled telomeric DNA elongation and increasing single strandedness. In tel1 mec1 cells, tlc1-476A telomerase was also capable of uncontrolled synthesis, but only after telomeres had shortened for >30 generations. These results show that, without Tel1p and Mec1p, telomerase is still active and can act in vivo when the telomere structure is disrupted by various means. Hence, a primary function of the ATM-family kinases in telomere maintenance is to act on the substrate of telomerase, the telomere, rather than to activate the enzymatic activity of telomerase (Chan, 2001).

Telomeres protect chromosome ends from fusing to double-stranded breaks (DSBs). Using a quantitative real-time PCR assay, it has been shown that nonhomologous end joining between a telomere and an inducible DSB is undetectable in wild-type cells, but occurs within a few hours of DSB induction in approximately 1/2000 genomes in telomerase-deficient cells and in >1/1000 genomes in telomerase-deficient cells also lacking the ATM homolog Tel1p. The fused telomeres contained very little telomeric DNA, suggesting that catastrophic telomere shortening preceded fusion. Lengthening of telomeres did not prevent such catastrophic telomere shortening and fusion events. Telomere-DSB fusion also occurred in cells containing a catalytically inactive telomerase and in tel1 mec1 cells where telomerase cannot elongate telomeres. Thus, telomerase and Tel1p function in telomere protection as well as in telomere elongation (Chan, 2003).

The phosphoinositide (PI)-3-kinase-related kinase (PIKK) family proteins Tel1p and Mec1p have been implicated in the telomere integrity of Saccharomyces cerevisiae. However, the mechanism of PIKK-mediated telomere length control remains unclear. HereTel1p and Mec1p are shown to be recruited to the telomeres at specific times in the cell cycle in a mutually exclusive manner. In particular, Mec1p interacts with the telomeres during late S phase and is associated preferentially with shortened telomeres. A model is proposed in which telomere integrity is maintained by the reciprocal association of PIKKs, and Mec1p acts as a sensor for structural abnormalities in the telomeres. This study suggests a mechanistic similarity between telomere length regulation and DNA double-strand break repair, both of which are achieved by the direct association of PIKKs (Takata, 2004).

Activation of ATM

The ATM protein kinase, mutations of which are associated with the human disease ataxia-telangiectasia, mediates responses to ionizing radiation in mammalian cells. ATM is held inactive in unirradiated cells as a dimer or higher-order multimer, with the kinase domain bound to a region surrounding serine 1981 that is contained within the previously described 'FAT' domain. Cellular irradiation induces rapid intermolecular autophosphorylation of serine 1981 that causes dimer dissociation and initiates cellular ATM kinase activity. Most ATM molecules in the cell are rapidly phosphorylated on this site after doses of radiation as low as 0.5 Gy, and binding of a phosphospecific antibody is detectable after the introduction of only a few DNA double-strand breaks in the cell. Activation of the ATM kinase seems to be an initiating event in cellular responses to irradiation, and the data indicate that ATM activation is not dependent on direct binding to DNA strand breaks, but may result from changes in the structure of chromatin (Bakkenist, 2003).

The telomeric protein TRF2 is required to prevent mammalian telomeres from activating DNA damage checkpoints. Overexpression of TRF2 affects the response of the ATM kinase to DNA damage. Overexpression of TRF2 abrogates the cell cycle arrest after ionizing radiation and diminishes several other readouts of the DNA damage response, including phosphorylation of Nbs1 (Drosophila homolog Nbs), induction of p53, and upregulation of p53 targets. TRF2 inhibits autophosphorylation of ATM on S1981, an early step in the activation of this kinase. A region of ATM containing S1981 directly interacts with TRF2 in vitro, and ATM immunoprecipitates contained TRF2. It is proposed that TRF2 has the ability to inhibit ATM activation at telomeres. Because TRF2 is abundant at chromosome ends but not elsewhere in the nucleus, this mechanism of checkpoint control could specifically block a DNA damage response at telomeres without affecting the surveillance of chromosome internal damage (Karlseder, 2004).

The complex containing the Mre11, Rad50, and Nbs1 proteins (MRN) is essential for the cellular response to DNA double-strand breaks, integrating DNA repair with the activation of checkpoint signaling through the protein kinase ATM. MRN stimulates the kinase activity of ATM in vitro toward its substrates p53, Chk2, and histone H2AX. MRN makes multiple contacts with ATM and appears to stimulate ATM activity by facilitating the stable binding of substrates. Phosphorylation of Nbs1 is critical for MRN stimulation of ATM activity toward Chk2, but not p53. Kinase-deficient ATM inhibits wild-type ATM phosphorylation of Chk2, consistent with the dominant-negative effect of kinase-deficient ATM in vivo (Lee, 2004).

The ataxia-telangiectasia mutated (ATM) kinase signals the presence of DNA double-strand breaks in mammalian cells by phosphorylating proteins that initiate cell-cycle arrest, apoptosis, and DNA repair. The Mre11-Rad50-Nbs1 (MRN) complex acts as a double-strand break sensor for ATM and recruits ATM to broken DNA molecules. Inactive ATM dimers were activated in vitro with DNA in the presence of MRN, leading to phosphorylation of the downstream cellular targets p53 and Chk2. ATM autophosphorylation is not required for monomerization of ATM by MRN. The unwinding of DNA ends by MRN is essential for ATM stimulation, consistent with the central role of single-stranded DNA as an evolutionarily conserved signal for DNA damage (Lee, 2005).

ATM has a central role in controlling the cellular responses to DNA damage. It and other phosphoinositide 3-kinase-related kinases (PIKKs) have giant helical HEAT repeat domains in their amino-terminal regions. The functions of these domains in PIKKs are not well understood. ATM activation in response to DNA damage appears to be regulated by the Mre11-Rad50-Nbs1 (MRN) complex, although the exact functional relationship between the MRN complex and ATM is uncertain. Two pairs of HEAT repeats in fission yeast ATM (Tel1) interact with an FXF/Y motif at the C terminus of Nbs1. This interaction resembles nucleoporin FXFG motif binding to HEAT repeats in importin-beta. Budding yeast Nbs1 (Xrs2) appears to have two FXF/Y motifs that interact with Tel1 (ATM). In Xenopus egg extracts, the C terminus of Nbs1 recruits ATM to damaged DNA, where it is subsequently autophosphorylated. This interaction is essential for ATM activation. A C-terminal 147-amino-acid fragment of Nbs1 that has the Mre11- and ATM-binding domains can restore ATM activation in an Nbs1-depleted extract. It is concluded that an interaction between specific HEAT repeats in ATM and the C-terminal FXF/Y domain of Nbs1 is essential for ATM activation. It is proposed that conformational changes in the MRN complex that occur upon binding to damaged DNA are transmitted through the FXF/Y-HEAT interface to activate ATM. This interaction also retains active ATM at sites of DNA damage (You, 2005).

The Atm protein kinase is central to the DNA double-strand break response in mammalian cells. After irradiation, dimeric Atm undergoes autophosphorylation at Ser 1981 and dissociates into active monomers. Atm activation is stimulated by expression of the Mre11/Rad50/nibrin complex. A C-terminal fragment of nibrin, containing binding sites for both Mre11 and Atm, is sufficient to provide this stimulatory effect in Nijmegen breakage syndrome (NBS) cells. To discriminate whether nibrin's role in Atm activation is to bind and translocate Mre11/Rad50 to the nucleus or to interact directly with Atm, an Mre11 transgene with a C-terminal NLS sequence was expressed in NBS fibroblasts. The Mre11-NLS protein complexes with Rad50, localizes to the nucleus in NBS fibroblasts, and associates with chromatin. However, Atm autophosphorylation is not stimulated in cells expressing Mre11-NLS, nor are downstream Atm targets phosphorylated. To determine whether nibrin-Atm interaction is necessary to stimulate Atm activation, nibrin transgenes lacking the Atm binding domain were expressed in NBS fibroblasts. The nibrin DeltaAtm protein interacted with Mre11/Rad50; however, Atm autophosphorylation is dramatically reduced after irradiation in NBS cells expressing the nibrin DeltaAtm transgenes relative to wild-type nibrin. These results indicate that nibrin plays an active role in Atm activation beyond translocating Mre11/Rad50 to the nucleus and that this function requires nibrin-Atm interaction (Cerosaletti, 2006).

DNA double-strand breaks (DSBs) trigger activation of the ATM protein kinase, which coordinates cell-cycle arrest, DNA repair and apoptosis. It is proposed that ATM activation by DSBs occurs in two steps. First, dimeric ATM is recruited to damaged DNA and dissociates into monomers. The Mre11-Rad50-Nbs1 complex (MRN) facilitates this process by tethering DNA, thereby increasing the local concentration of damaged DNA. Notably, increasing the concentration of damaged DNA bypasses the requirement for MRN, and ATM monomers generated in the absence of MRN are not phosphorylated on Ser1981. Second, the ATM-binding domain of Nbs1 is required and sufficient to convert unphosphorylated ATM monomers into enzymatically active monomers in the absence of DNA. This model clarifies the mechanism of ATM activation in normal cells and explains the phenotype of cells from patients with ataxia telangiectasia-like disorder and Nijmegen breakage syndrome (Dupre, 2006).

In response to DNA damage, cells undergo either cell-cycle arrest or apoptosis, depending on the extent of damage and the cell's capacity for DNA repair. Cell-cycle arrest induced by double-stranded DNA breaks depends on activation of the ataxia-telangiectasia (ATM) protein kinase, which phosphorylates cell-cycle effectors such as Chk2 and p53 to inhibit cell-cycle progression. ATM is recruited to double-stranded DNA breaks by a complex of sensor proteins, including Mre11/Rad50/Nbs1, resulting in autophosphorylation, monomerization, and activation of ATM kinase. In characterizing Aven protein, a previously reported apoptotic inhibitor, it was found that Aven can function as an ATM activator to inhibit G2/M progression. Aven bound to ATM and Aven overexpressed in cycling Xenopus egg extracts prevents mitotic entry and induces phosphorylation of ATM and its substrates. Immunodepletion of endogenous Aven allows mitotic entry even in the presence of damaged DNA, and RNAi-mediated knockdown of Aven in human cells prevents autophosphorylation of ATM at an activating site (S1981) in response to DNA damage. Interestingly, Aven is also a substrate of the ATM kinase. Mutation of ATM-mediated phosphorylation sites on Aven reduces its ability to activate ATM, suggesting that Aven activation of ATM after DNA damage is enhanced by ATM-mediated Aven phosphorylation. These results identify Aven as a new ATM activator and describe a positive feedback loop operating between Aven and ATM. In aggregate, these findings place Aven, a known apoptotic inhibitor, as a critical transducer of the DNA-damage signal (Guo, 2008).

ATM structure/function studies

The ATM protein has been implicated in pathways controlling cell cycle checkpoints, radiosensitivity, genetic instability, and aging. Expression of ATM fragments containing a leucine zipper motif in a human tumor cell line abrogates the S-phase checkpoint after ionizing irradiation and enhances radiosensitivity and chromosomal breakage. These fragments did not abrogate irradiation-induced G1 or G2 checkpoints, suggesting that cell cycle checkpoint defects alone cannot account for chromosomal instability in ataxia telangiectasia (AT) cells. Expression of the carboxy-terminal portion of ATM, which contains the PI-3 kinase domain, complements radiosensitivity and the S-phase checkpoint and reduces chromosomal breakage after irradiation in AT cells. These observations suggest that ATM function is dependent on interactions with itself or other proteins through the leucine zipper region and that the PI-3 kinase domain contains much of the significant activity of ATM (Morgan, 1997).

ATM phosphorylates p53 protein in response to ionizing radiation, but the complex phenotype of AT cells suggests that it must have other cellular substrates as well. To identify substrates for ATM and the related kinases ATR (ATM and Rad3 related) and DNA-PK, in vitro kinase assays were optimized and a rapid peptide screening method was developed to determine general phosphorylation consensus sequences. ATM and ATR require Mn(2+), but not DNA ends or Ku proteins, for optimal in vitro activity while DNA-PKCs require Mg(2+), DNA ends, and Ku proteins. From p53 peptide mutagenesis analysis, it was found that the sequence S/TQ is a minimal essential requirement for all three kinases. In addition, hydrophobic amino acids and negatively charged amino acids immediately NH(2)-terminal to serine or threonine are positive determinants and positively charged amino acids in the region are negative determinants for substrate phosphorylation. A general phosphorylation consensus sequence for ATM was determined and putative in vitro targets were identified by using glutathione S-transferase peptides as substrates. Putative ATM in vitro targets include p95/nibrin, Mre11, Brca1, Rad17, PTS, WRN, and ATM (S440) itself. Brca2, phosphatidylinositol 3-kinase, and DNA-5B peptides were phosphorylated specifically by ATR, and DNA Ligase IV is a specific in vitro substrate of DNA-PK (Kim, 1999).

ATM targets Chk1 and Chk2

The protein kinase Chk2, the mammalian homolog of the budding yeast Rad53 and fission yeast Cds1 checkpoint kinases, is phosphorylated and activated in response to DNA damage by ionizing radiation (IR), UV irradiation, and replication blocks by hydroxyurea (HU). Phosphorylation and activation of Chk2 are ATM dependent in response to IR, whereas Chk2 phosphorylation is ATM-independent when cells are exposed to UV or HU. ATM phosphorylates in vitro the Ser-Gln/Thr-Gln (SQ/TQ) cluster domain (SCD) on Chk2, which contains seven SQ/TQ motifs, and Thr68 is the major in vitro phosphorylation site by ATM. ATM- and Rad3-related also phosphorylates Thr68 in addition to Thr26 and Ser50, which are not phosphorylated to a significant extent by ATM in vitro. In vivo, Thr68 is phosphorylated in an ATM-dependent manner in response to IR, but not in response to UV or HU. Substitution of Thr68 with Ala reduces the extent of phosphorylation and activation of Chk2 in response to IR, and mutation of all seven SQ/TQ motifs blocks all phosphorylation and activation of Chk2 after IR. These results suggest that in vivo, Chk2 is directly phosphorylated by ATM in response to IR and that Chk2 is regulated by phosphorylation of the SCD (Matsuoka, 2000).

ATM is necessary for phosphorylation and activation of Cds1/Chk2 in vivo and can phosphorylate Cds1 in vitro, although evidence is lacking that the sites phosphorylated by ATM are required for activation. This study shows that threonine 68 of Cds1 is the preferred site of phosphorylation by ATM in vitro, and is the principal irradiation-induced site of phosphorylation in vivo. The importance of this phosphorylation site is demonstrated by the failure of a mutant, non-phosphorylatable form of Cds1 to be fully activated, and by its reduced ability to induce G1 arrest in response to ionizing radiation (Melchionna, 2000).

In response to ionizing radiation (IR), the tumor suppressor p53 is stabilized and promotes either cell cycle arrest or apoptosis. Chk2 activated by IR contributes to this stabilization, possibly by direct phosphorylation. Like p53, Chk2 is mutated in patients with Li-Fraumeni syndrome. Since the ATM gene is required for IR-induced activation of Chk2, it has been assumed that ATM and Chk2 act in a linear pathway leading to p53 activation. To clarify the role of Chk2 in tumorigenesis, gene-targeted Chk2-deficient mice were generated. Unlike ATM(-/-) and p53(-/-) mice, Chk2(-/-) mice do not spontaneously develop tumors, although Chk2 does suppress 7,12-dimethylbenzanthracene-induced skin tumors. Tissues from Chk2(-/-) mice, including those from the thymus, central nervous system, fibroblasts, epidermis, and hair follicles, show significant defects in IR-induced apoptosis or impaired G(1)/S arrest. Quantitative comparison of the G(1)/S checkpoint, apoptosis, and expression of p53 proteins in Chk2(-/-) versus ATM(-/-) thymocytes suggests that Chk2 can regulate p53-dependent apoptosis in an ATM-independent manner. IR-induced apoptosis is restored in Chk2(-/-) thymocytes by reintroduction of the wild-type Chk2 gene but not by a Chk2 gene in which the sites phosphorylated by ATM and ataxia telangiectasia and rad3⁺ related (ATR) are mutated to alanine. ATR may thus selectively contribute to p53-mediated apoptosis. These data indicate that distinct pathways regulate the activation of p53 leading to cell cycle arrest or apoptosis (Hirao, 2002).

In mammals, the ATM and ATR protein kinases function as critical regulators of the cellular DNA damage response. The checkpoint functions of ATR and ATM are mediated, in part, by a pair of checkpoint effector kinases termed Chk1 and Chk2. In mammalian cells, evidence has been presented that Chk1 is devoted to the ATR signaling pathway and is modified by ATR in response to replication inhibition and UV-induced damage, whereas Chk2 functions primarily through ATM in response to ionizing radiation (IR), suggesting that Chk2 and Chk1 might have evolved to channel the DNA damage signal from ATM and ATR, respectively. The ATR-Chk1 and ATM-Chk2 pathways are not parallel branches of the DNA damage response pathway but instead show a high degree of cross-talk and connectivity. ATM does in fact signal to Chk1 in response to IR. Phosphorylation of Chk1 on Ser-317 in response to IR is ATM-dependent. Functional NBS1 is required for phosphorylation of Chk1, indicating that NBS1 might facilitate the access of Chk1 to ATM at the sites of DNA damage. Abrogation of Chk1 expression by RNA interference results in defects in IR-induced S and G(2)/M phase checkpoints; however, the overexpression of phosphorylation site mutant (S317A, S345A or S317A/S345A double mutant) Chk1 fails to interfere with these checkpoints. Surprisingly, the kinase-dead Chk1 (D130A) also fails to abrogate the S and G(2) checkpoint through any obvious dominant negative effect toward endogenous Chk1. Therefore, further studies will be required to assess the contribution made by phosphorylation events to Chk1 regulation. Overall, the data presented in the study challenge the model in which Chk1 functions downstream from ATR only and does indicate that ATM signals to Chk1. In addition, this study also demonstrates that Chk1 is essential for IR-induced inhibition of DNA synthesis and the G(2)/M checkpoint (Gatei, 2003).

Eukaryotic cells activate an evolutionarily conserved set of proteins that rapidly induce cell cycle arrest to prevent replication or segregation of damaged DNA before repair is completed. In response to ionizing radiation (IR), the cell cycle checkpoint kinase, Chk2 (hCds1), is phosphorylated and activated in an ataxia telangiectasia mutated (ATM)-dependent manner. The ATM protein kinase directly phosphorylates T68 within the SQ/TQ-rich domain of Chk2 in vitro and T68 is phosphorylated in vivo in response to IR in an ATM-dependent manner. Furthermore, phosphorylation of T68 was required for full activation of Chk2 after IR. Together, these data are consistent with the model that ATM directly phosphorylates Chk2 in vivo and that this event contributes to the activation of Chk2 in irradiated cells (Ahn, 2000).

When exposed to ionizing radiation (IR), eukaryotic cells activate checkpoint pathways to delay the progression of the cell cycle. Defects in the IR-induced S-phase checkpoint cause 'radioresistant DNA synthesis', a phenomenon that has been identified in cancer-prone patients suffering from ataxia-telangiectasia, a disease caused by mutations in the ATM gene. The Cdc25A phosphatase activates the cyclin-dependent kinase 2 (Cdk2) needed for DNA synthesis, but becomes degraded in response to DNA damage or stalled replication. A functional link is reported between ATM, the checkpoint signalling kinase Chk2/Cds1 (Chk2) and Cdc25A, and this mechanism is implicated in controlling the S-phase checkpoint. IR-induced destruction of Cdc25A requires both ATM and the Chk2-mediated phosphorylation of Cdc25A on serine 123. An IR-induced loss of Cdc25A protein prevents dephosphorylation of Cdk2 and leads to a transient blockade of DNA replication. Tumor-associated Chk2 alleles cannot bind or phosphorylate Cdc25A, and cells expressing these Chk2 alleles or elevated Cdc25A, or a Cdk2 mutant unable to undergo inhibitory phosphorylation (Cdk2AF) all fail to inhibit DNA synthesis when irradiated. These results support Chk2 as a candidate tumor suppressor, and identify the ATM-Chk2-Cdc25A-Cdk2 pathway as a genomic integrity checkpoint that prevents radioresistant DNA synthesis (Falck, 2001).

The Chk2 Ser/Thr kinase plays crucial, evolutionarily conserved roles in cellular responses to DNA damage. Identification of two pro-oncogenic mutations within the Chk2 FHA domain has highlighted its importance for Chk2 function in checkpoint activation. The X-ray structure of the Chk2 FHA domain in complex with an in vitro selected phosphopeptide motif reveals the determinants of binding specificity and shows that both mutations are remote from the peptide binding site. The Chk2 FHA domain mediates ATM-dependent Chk2 phosphorylation and targeting of Chk2 to in vivo binding partners such as BRCA1 through either or both of two structurally distinct mechanisms. Although phospho-dependent binding is important for Chk2 activity, previously uncharacterized phospho-independent FHA domain interactions appear to be the primary target of oncogenic lesions (Li, 2002).

Timing of DNA replication initiation is dependent on S-phase-promoting kinase (SPK) activity at discrete origins and the simultaneous function of many replicons. DNA damage prevents origin firing through the ATM- and ATR-dependent inhibition of Cdk2 and Cdc7 SPKs. Modulation of ATM- and ATR-signalling pathways controls origin firing in the absence of DNA damage. Inhibition of ATM and ATR with caffeine or specific neutralizing antibodies, or upregulation of Cdk2 or Cdc7, promotes rapid and synchronous origin firing; conversely, inhibition of Cdc25A slows DNA replication. Cdk2 was in equilibrium between active and inactive states, and the concentration of replication protein A (RPA)-bound single-stranded DNA (ssDNA) correlated with Chk1 activation and inhibition of origin firing. Furthermore, ATM was transiently activated during ongoing replication. It is proposed that ATR and ATM regulate SPK activity through a feedback mechanism originating at active replicons. These observations establish that ATM- and ATR-signalling pathways operate during an unperturbed cell cycle to regulate initiation and progression of DNA synthesis, and are therefore poised to halt replication in the presence of DNA damage (Shechter, 2004).

ATM targets Rad9

ATM is a Ser/Thr kinase involved in cell cycle checkpoints and DNA repair. Human Rad9 (hRad9) is the homolog of Schizosaccharomyces pombe Rad9 protein that plays a critical role in cell cycle checkpoint control. To examine the potential signaling pathway linking ATM and hRad9, the modification of hRad9 in response to DNA damage was investigated. hRad9 protein is constitutively phosphorylated in undamaged cells and undergoes hyperphosphorylation upon treatment with ionizing radiation (IR), ultraviolet light (UV), and hydroxyurea (HU). Interestingly, hyperphosphorylation of hRad9 induced by IR is dependent on ATM. Ser(272) of hRad9 is phosphorylated directly by ATM in vitro. Furthermore, hRad9 is phosphorylated on Ser(272) in response to IR in vivo, and this modification is delayed in ATM-deficient cells. Expression of hRad9 S272A mutant protein in human lung fibroblast VA13 cells disturbs IR-induced G(1)/S checkpoint activation and increases cellular sensitivity to IR. Together, these results suggest that the ATM-mediated phosphorylation of hRad9 is required for IR-induced checkpoint activation (Chen, 2001).

To gain insight into the function and organization of proteins assembled on the DNA in response to genotoxic insult, the phosphorylation of the Schizosaccharomyces pombe PCNA-like checkpoint protein Rad9 was investigated. C-terminal T412/S423 phosphorylation of Rad9 by Rad3^ATR occurs in S phase without replication stress. Rad3^ATR and Tel1^ATM phosphorylate these same residues, plus additional ones, in response to DNA damage. In S phase and after damage, only Rad9 phosphorylated on T412/S423, but not unphosphorylated Rad9, associates with a two-BRCT-domain region of the essential Rad4^TOPBP1 protein. Rad9-Rad4^TOPBP1 interaction is required to activate the Chk1 damage checkpoint but not the Cds1 replication checkpoint. When the Rad9-T412/S423 are phosphorylated, Rad4^TOPBP1 coprecipitates with Rad3^ATR, suggesting that phosphorylation coordinates formation of an active checkpoint complex (Furuya, 2004).

Most of the proteins involved in the DNA damage and DNA replication checkpoint have been identified, and the majority are highly conserved through evolution. Many features of the checkpoint pathways remain unexplained, including their apparent complexity and the fact that many of the same proteins participate in both the DNA damage and DNA replication responses. The data presented in this study begin to uncover the molecular organization of the checkpoint proteins following their recruitment to sites of DNA damage or collapsed DNA replication forks. It is suggested that one of the reasons for the apparent complexity of the system is because it allows cells to distinguish between similar biochemical consequences of DNA damage (such as ssDNA-RPA complexes) that occurs in distinct circumstances (such as induced damage in G2 and collapsed replication forks). It is important to make these distinctions because different signaling responses to the cell cycle and the DNA repair apparatus will be appropriate in each case. The data suggest that the Rad3^ATR-dependent phosphorylation of Rad9 promotes association between Rad9 and Rad4^TOPBP1 through phospho-specific BRCT-domain interactions during unperturbed S phase, and that this helps cells distinguish collapsed forks from DNA damage in G2 cells. It is intriguing that a phospho-specific BRCT-mediated interaction between BRCA1 and BACH1 in human cells is promoted by cyclin-dependent kinase activity against BACH1 in G2 and also by the G2 checkpoint. Together, these observations suggest that a combination of phosphorylation events can orchestrate the organization of the checkpoint apparatus before it is activated and that, upon activation, the consequent phospho-specific protein interactions dictate the downstream consequences of this activation (Furuya, 2004).

The Rad9-Hus1-Rad1 (9-1-1) clamp activates checkpoint signaling via TopBP1

DNA replication stress triggers the activation of Checkpoint Kinase 1 (Chk1) in a pathway that requires the independent chromatin loading of the ATRIP-ATR (ATR-interacting protein/ATM [ataxia-telangiectasia mutated]-Rad3-related kinase) complex and the Rad9-Hus1-Rad1 (9-1-1) clamp (See Drosophila Hus1). Rad9’s role in Chk1 activation is to bind TopBP1, which stimulates ATR-mediated Chk1 phosphorylation via TopBP1’s activation domain (AD), a domain that binds and activates ATR. Notably, fusion of the AD to proliferating cell nuclear antigen (PCNA) or histone H2B bypasses the requirement for the 9-1-1 clamp, indicating that the 9-1-1 clamp’s primary role in activating Chk1 is to localize the AD to a stalled replication fork (Delacroix, 2007).

Genotoxic damage activates conserved checkpoint signaling pathways that maintain genomic stability by regulating cell cycle progression, triggering apoptosis, and influencing DNA repair. One pathway that is potently activated by replication stress leads to activation of Checkpoint Kinase 1 (Chk1), which promotes cell survival by blocking the firing of replication origins, preventing entry into mitosis, stabilizing stalled replication forks, and facilitating DNA repair. This pathway is initiated when the replicative DNA polymerases stall and large tracts of single-stranded DNA are created by the uncoupling of the replicative helicase from the advancing replication fork. The single-stranded DNA is then coated by replication protein A (RPA), which signals the independent recruitment of two checkpoint complexes: the ataxia-telangiectasia mutated (ATM)-Rad3-related kinase-ATR-interacting protein (ATR-ATRIP) complex and the Rad9-Hus1-Rad1 (9-1-1) complex. The ATRIP-ATR complex is bound to DNA by a direct interaction between ATRIP and RPA. In contrast, loading of the 9-1-1 complex requires several steps. First, DNA polymerase is recruited, which in turn recruits the clamp loader, Rad17-replication factor C (RFC). Second, the Rad17-RFC then loads the proliferating cell nuclear antigen (PCNA)-like 9-1-1 complex onto chromatin in a reaction that is analogous to the loading of PCNA onto sites of DNA replication. Although the binding of the ATRIP-ATR complex and the loading of the 9-1-1 complex occur independently of one another, both events are essential for optimal ATR-mediated Chk1 phosphorylation and activation (Delacroix, 2007 and references therein).

Despite the tremendous progress that has been made in deciphering the biochemical functions of the 9-1-1 complex and the in-depth understanding of the signals that lead to the loading of the 9-1-1 clamp, it has remained unclear how the chromatin-bound 9-1-1 complex initiates and propagates the Chk1-activating signal. Several studies have demonstrated that Rad9 orthologs in Schizosaccharomyces pombe, Saccharomyces cerevisiae, and humans interact with their respective TopBP1 orthologs (Cut4, Dpb11, and TopBP1). However, the significance of the Rad9-TopBP1 interaction in 9-1-1 function has not been explored. This study shows that the role of the 9-1-1 clamp is to recruit TopBP1, which then triggers ATR-mediated Chk1 phosphorylation. Thus, TopBP1 is a molecular bridge that links the independently recruited 9-1-1 and ATRIP-ATR complexes, leading to checkpoint activation (Delacroix, 2007).

ATM targets the MCM complex

The minichromosome maintenance (MCM) 2-7 helicase complex functions to initiate and elongate replication forks. Cell cycle checkpoint signaling pathways regulate DNA replication to maintain genomic stability. Four lines of evidence are described that ATM/ATR-dependent (ataxia-telangiectasia-mutated/ATM- and Rad3-related) checkpoint pathways are directly linked to three members of the MCM complex. First, ATM phosphorylates MCM3 on S535 in response to ionizing radiation. Second, ATR phosphorylates MCM2 on S108 in response to multiple forms of DNA damage and stalling of replication forks. Third, ATR-interacting protein (ATRIP)-ATR interacts with MCM7. Fourth, reducing the amount of MCM7 in cells disrupts checkpoint signaling and causes an intra-S-phase checkpoint defect. Thus, the MCM complex is a platform for multiple DNA damage-dependent regulatory signals that control DNA replication (Cortez, 2004).

In vertebrates, ATM and ATR are critical regulators of checkpoint responses to damaged and incompletely replicated DNA. These checkpoint responses involve the activation of signaling pathways that inhibit the replication of chromosomes with DNA lesions. A cDNA has been isolated encoding a full-length version of Xenopus ATM. Using antibodies against the regulatory domain of ATM, the essential replication protein Mcm2 has been identified as an ATM-binding protein in Xenopus egg extracts. Xenopus Mcm2 undergoes phosphorylation on serine 92 (S92) in response to the presence of double-stranded DNA breaks or DNA replication blocks in egg extracts. This phosphorylation involves both ATM and ATR, but the relative contribution of each kinase depends upon the checkpoint-inducing DNA signal. Furthermore, both ATM and ATR phosphorylate Mcm2 directly on S92 in cell-free kinase assays. Immunodepletion of both ATM and ATR from egg extracts abrogates the checkpoint response that blocks chromosomal DNA replication in egg extracts containing double-stranded DNA breaks. These experiments indicate that ATM and ATR phosphorylate the functionally critical replication protein Mcm2 during both DNA damage and replication checkpoint responses in Xenopus egg extracts (Yoo, 2004).

ATM associates with and phosphorylates p53: ATM and a spindle assembly checkpoint

The human genetic disorder ataxia-telangiectasia (AT) is characterized by immunodeficiency, progressive cerebellar ataxia, radiosensitivity, cell cycle checkpoint defects and cancer predisposition. The gene mutated in this syndrome, ATM (for AT mutated), encodes a protein containing a phosphatidyl-inositol 3-kinase (PI-3 kinase)-like domain. ATM also contains a proline-rich region and a leucine zipper, both of which implicate this protein in signal transduction. The proline-rich region has been shown to bind to the SH3 domain of c-Abl, which facilitates its phosphorylation and activation by ATM. AT cells are defective in the G1/S checkpoint activated after radiation damage and this defect is attributable to a defective p53 signal transduction pathway. There is a direct interaction between ATM and p53 involving two regions in ATM, one at the amino terminus and the other at the carboxy terminus, corresponding to the PI-3 kinase domain. Recombinant ATM protein phosphorylates p53 on serine 15 near the N terminus. Furthermore, ectopic expression of ATM in AT cells restores normal ionizing radiation (IR)-induced phosphorylation of p53, whereas expression of ATM antisense RNA in control cells abrogates the rapid IR-induced phosphorylation of p53 on serine 15. These results demonstrate that ATM can bind p53 directly and is responsible for its serine 15 phosphorylation, thereby contributing to the activation and stabilization of p53 during the IR-induced DNA damage response (Khanna, 1998).

The p53 oncosuppressor associates to centrosomes in mitosis and this association is disrupted by treatments with microtubule-depolymerizing agents. ATM, an upstream activator of p53 after DNA damage, is essential for p53 centrosomal localization and is required for the activation of the postmitotic checkpoint after spindle disruption. In mitosis, p53 failed to associate with centrosomes in two ATM-deficient, ataxiatelangiectasia-derived cell lines. Wild-type ATM gene transfer reestablished the centrosomal localization of p53 in these cells. Furthermore, wild-type p53 protein, but not the p53-S15A mutant, not phosphorylatable by ATM, localized at centrosomes when expressed in p53-null K562 cells. Finally, Ser15 phosphorylation of endogenous p53 was detected at centrosomes upon treatment with phosphatase inhibitors, suggesting that a p53 dephosphorylation step at the centrosome contributes to sustain the cell cycle program in cells with normal mitotic spindles. When dissociated from the centrosomes by treatments with spindle inhibitors, p53 remains phosphorylated at Ser15. AT cells, which are unable to phosphorylate p53, do not undergo postmitotic proliferation arrest after nocodazole block and release. These data demonstrate that ATM is required for p53 localization at the centrosome and support the existence of a surveillance mechanism for inhibiting DNA reduplication downstream of the spindle assembly checkpoint (Tritarelli, 2004).

ATM-mediated stabilization of hMutL DNA mismatch repair proteins augments p53 activation during DNA damage

Human DNA mismatch repair (MMR) proteins correct DNA errors and regulate cellular response to DNA damage by signaling apoptosis. Mutations of MMR genes result in genomic instability and cancer development. Nonetheless, how MMR proteins are regulated has not yet been determined. While hMLH1, hPMS2, and hMLH3 are known to participate in MMR, the function of another member of MutL-related proteins, hPMS1, remains unclear. DNA damage induces the accumulation of hPMS1, hPMS2, and hMLH1 through ataxia-telangiectasia-mutated (ATM)-mediated protein stabilization. The subcellular localization of PMS proteins is also regulated during DNA damage, which induces nuclear localization of hPMS1 and hPMS2 in an hMLH1-dependent manner. The induced levels of hMLH1 and hPMS1 are important for the augmentation of p53 phosphorylation by ATM in response to DNA damage. These observations identify hMutL proteins as regulators of p53 response and demonstrate for the first time a function of hMLH1-hPMS1 complex in controlling the DNA damage response (Luo, 2004).

The combined status of ATM and p53 link tumor development with therapeutic response

While the contribution of specific tumor suppressor networks to cancer development has been the subject of considerable recent study, it remains unclear how alterations in these networks are integrated to influence the response of tumors to anti-cancer treatments. This study shows that mechanisms commonly used by tumors to bypass early neoplastic checkpoints ultimately determine chemotherapeutic response and generate tumor-specific vulnerabilities that can be exploited with targeted therapies. Specifically, evaluation of the combined status of ATM and p53, two commonly mutated tumor suppressor genes, can help to predict the clinical response to genotoxic chemotherapies. This study shows that in p53-deficient settings, suppression of ATM dramatically sensitizes tumors to DNA-damaging chemotherapy, whereas, conversely, in the presence of functional p53, suppression of ATM or its downstream target Chk2 actually protects tumors from being killed by genotoxic agents. Furthermore, ATM-deficient cancer cells display strong nononcogene addiction to DNA-PKcs for survival after DNA damage, such that suppression of DNA-PKcs in vivo resensitizes inherently chemoresistant ATM-deficient tumors to genotoxic chemotherapy. Thus, the specific set of alterations induced during tumor development plays a dominant role in determining both the tumor response to conventional chemotherapy and specific susceptibilities to targeted therapies in a given malignancy (Jiang, 2009).

Chk1 suppresses Caspase-2 apoptotic response to DNA damage that bypasses p53, Bcl-2, and Caspase-3 but requires ATM

Evasion of DNA damage-induced cell death, via mutation of the p53 tumor suppressor or overexpression of prosurvival Bcl-2 family proteins, is a key step toward malignant transformation and therapeutic resistance. Depletion or acute inhibition of checkpoint kinase 1 (Chk1) is sufficient to restore γ-radiation-induced apoptosis in p53 mutant zebrafish embryos. Surprisingly, caspase-3 is not activated prior to DNA fragmentation, in contrast to classical intrinsic or extrinsic apoptosis. Rather, an alternative apoptotic program is engaged that cell autonomously requires atm (ataxia telangiectasia mutated), atr (ATM and Rad3-related) and caspase-2, and is not affected by p53 loss or overexpression of bcl-2/xl. Similarly, Chk1 inhibitor-treated human tumor cells hyperactivate ATM, ATR, and caspase-2 after γ-radiation and trigger a caspase-2-dependent apoptotic program that bypasses p53 deficiency and excess Bcl-2. The evolutionarily conserved 'Chk1-suppressed' pathway defines a novel apoptotic process, whose responsiveness to Chk1 inhibitors and insensitivity to p53 and BCL2 alterations have important implications for cancer therapy (Sidi, 2008).

ATM targets the transcriptional cofactor Strap

The related kinases ATM and ATR phosphorylate a limited number of downstream protein targets in response to DNA damage. A new pathway is described in which ATM kinase signals the DNA damage response by targeting the transcriptional cofactor Strap. ATM phosphorylates Strap at a serine residue, stabilizing nuclear Strap and facilitating formation of a stress-responsive co-activator complex. Strap activity enhances p53 acetylation, and augments the response to DNA damage. Strap remains localized in the cytoplasm in cells derived from ataxia telangiectasia individuals with defective ATM, as well as in cells expressing a Strap mutant that cannot be phosphorylated by ATM. Targeting Strap to the nucleus reinstates protein stabilization and activates the DNA damage response. These results indicate that the nuclear accumulation of Strap is a critical regulator in the damage response, and argue that this function can be assigned to ATM through the DNA damage-dependent phosphorylation of Strap (Demonacos, 2004).

ATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway

The rare diseases ataxia-telangiectasia (AT), caused by mutations in the ATM gene, and Nijmegen breakage syndrome (NBS), with mutations in the p95/nbs1 gene, share a variety of phenotypic abnormalities such as chromosomal instability, radiation sensitivity and defects in cell-cycle checkpoints in response to ionizing radiation. The ATM gene encodes a protein kinase that is activated by ionizing radiation or radiomimetic drugs, whereas p95/nbs1 is part of a protein complex that is involved in responses to DNA double-strand breaks. Because of the similarities between AT and NBS, the functional interactions between ATM and p95/nbs1 were evaluated. Activation of the ATM kinase by ionizing radiation and induction of ATM-dependent responses in NBS cells indicates that p95/nbs1 may not be required for signalling to ATM after ionizing radiation. However, p95/nbs1 is phosphorylated on serine 343 in an ATM-dependent manner in vitro and in vivo after ionizing radiation. A p95/nbs1 construct mutated at the ATM phosphorylation site abrogates an S-phase checkpoint induced by ionizing radiation in normal cells and fails to compensate for this functional deficiency in NBS cells. These observations link ATM and p95/nbs1 in a common signalling pathway and provide an explanation for phenotypic similarities in these two diseases (Lim, 2000).

Inhibition of Polo-like kinase-1 by DNA damage occurs in an ATM- or ATR-dependent fashion

Polo-like kinases play multiple roles in different phases of mitosis. The mammalian polo-like kinase, Plk1, is inhibited in response to DNA damage and this inhibition may lead to cell cycle arrests at multiple points in mitosis. The role of the checkpoint kinases ATM and ATR in DNA damage-induced inhibition of Plk1 has been investigated. Inhibition of Plk1 kinase activity is efficiently blocked by the radio-sensitizing agent caffeine. Using ATM(-/-) cells it has been shown that under certain circumstances, inhibition of Plk1 by DNA-damaging agents critically depends on ATM. In addition, UV radiation also causes inhibition of Plk1, and evidence is presented that this inhibition is mediated by ATR. Taken together, these data demonstrate that ATM and ATR can regulate Plk1 kinase activity in response to a variety of DNA-damaging agents (van Vugt, 2001).

Phosphorylation of SMC1 is a critical downstream event in the ATM-NBS1-BRCA1 pathway

Structural maintenance of chromosomes (SMC) proteins (SMC1, SMC3) are evolutionarily conserved chromosomal proteins that are components of the cohesin complex, necessary for sister chromatid cohesion. These proteins may also function in DNA repair. SMC1 is a component of the DNA damage response network that functions as an effector in the ATM/NBS1-dependent S-phase checkpoint pathway. SMC1 associates with BRCA1 and is phosphorylated in response to IR in an ATM- and NBS1-dependent manner. Using mass spectrometry, it has been established that ATM phosphorylates S957 and S966 of SMC1 in vivo. Phosphorylation of S957 and/or S966 of SMC1 is required for activation of the S-phase checkpoint in response to IR. The phosphorylation of NBS1 (Nijmegen breakage syndrome gene product is a part of the hMre11 complex, a central player associated with double-strand break repair) by ATM is required for the phosphorylation of SMC1, establishing the role of NBS1 as an adaptor in the ATM/NBS1/SMC1 pathway. The ATM/CHK2/CDC25A pathway is also involved in the S-phase checkpoint activation, but this pathway is intact in NBS cells. These results indicate that the ATM/NBS1/SMC1 pathway is a separate branch of the S-phase checkpoint pathway, distinct from the ATM/CHK2/CDC25A branch. Therefore, this work establishes the ATM/NBS1/SMC1 branch, and provides a molecular basis for the S-phase checkpoint defect in NBS cells (Yazdi, 2002).

Structural maintenance of chromosomes (SMC) proteins play important roles in sister chromatid cohesion, chromosome condensation, sex-chromosome dosage compensation, and DNA recombination and repair. Protein complexes containing heterodimers of the Smc1 and Smc3 proteins have been implicated specifically in both sister chromatid cohesion and DNA recombination. The protein kinase Atm phosphorylates Smc1 protein after ionizing irradiation. Atm phosphorylates Smc1 on serines 957 and 966 in vitro and in vivo, and expression of an Smc1 protein mutated at these phosphorylation sites abrogates the ionizing irradiation-induced S phase cell cycle checkpoint. Optimal phosphorylation of these sites in Smc1 after ionizing irradiation also requires the presence of the Atm substrates Nbs1 and Brca1. These same sites in Smc1 are phosphorylated after treatment with UV irradiation or hydroxyurea in an Atm-independent manner, thus demonstrating that another kinase must be involved in responses to these cellular stresses. Yeast containing hypomorphic mutations in SMC1 and human cells overexpressing Smc1 mutated at both of these phosphorylation sites exhibit decreased survival following ionizing irradiation. These results demonstrate that Smc1 participates in cellular responses to DNA damage and link Smc1 to the Atm signal transduction pathway (Kim, 2002).

The ATM protein kinase is activated by intermolecular autophosphorylation in response to DNA damage and initiates cellular signaling pathways that facilitate cell survival and reduce chromosomal breakage. NBS1 and BRCA1 are required for the recruitment of previously activated ATM to the sites of DNA breaks after ionizing irradiation, and this recruitment is required for the phosphorylation of structural maintenance of chromosome protein 1 (SMC1) by ATM. To explore the functional importance of SMC1 phosphorylation, murine cells were generated, in which the two damage-induced phosphorylation sites in SMC1 are mutated. Although these cells demonstrate normal phosphorylation and focus formation of ATM, NBS1, and BRCA1 proteins after IR, they exhibit a defective S-phase checkpoint, decreased survival, and increased chromosomal aberrations after DNA damage. These observations suggest that many of the abnormal stress responses seen in cells lacking ATM, NBS1, or BRCA1 result from a failure of ATM migration to sites of DNA breaks and a resultant lack of SMC1 phosphorylation (Kitagawa, 2004).

BRCA1 acts in concert with ATM to regulate c-Abl tyrosine kinase activity

BRCA1 plays an important role in mechanisms of response to double-strand breaks, participating in genome surveillance, DNA repair, and cell cycle checkpoint arrests. This study identifies a constitutive BRCA1-c-Abl complex and evidence is provided for a direct interaction between the PXXP motif in the C terminus of BRCA1 and the SH3 domain of c-Abl. Following exposure to ionizing radiation (IR), the BRCA1-c-Abl complex is disrupted in an ATM-dependent manner, which correlates temporally with ATM-dependent phosphorylation of BRCA1 and ATM-dependent enhancement of the tyrosine kinase activity of c-Abl. The BRCA1-c-Abl interaction is affected by radiation-induced modification to both BRCA1 and c-Abl. The C terminus of BRCA1 is phosphorylated by c-Abl in vitro. In vivo, BRCA1 is phosphorylated at tyrosine residues in an ATM-dependent, radiation-dependent manner. Tyrosine phosphorylation of BRCA1, however, is not required for the disruption of the BRCA1-c-Abl complex. BRCA1-mutated cells exhibit constitutively high c-Abl kinase activity that is not further increased on exposure to IR. A model is suggested in which BRCA1 acts in concert with ATM to regulate c-Abl tyrosine kinase activity (Foray, 2002).

Human Rif1, ortholog of a yeast telomeric protein, is regulated by ATM and 53BP1 and functions in the S-phase checkpoint

The function has been examined of the human ortholog of Saccharomyces cerevisiae Rif1 (Rap1-interacting factor 1). Yeast Rif1 associates with telomeres and regulates their length. In contrast, human Rif1 does not accumulate at functional telomeres, but localizes to dysfunctional telomeres and to telomeric DNA clusters in ALT cells, a pattern of telomere association typical of DNA-damage-response factors. After induction of double-strand breaks (DSBs), Rif1 forms foci that colocalize with other DNA-damage-response factors. This response is strictly dependent on ATM and 53BP1, but not affected by diminished function of ATR (ATM- and Rad3-related kinase), BRCA1, Chk2, Nbs1, and Mre11. Rif1 inhibition results in radiosensitivity and a defect in the intra-S-phase checkpoint. The S-phase checkpoint phenotype is independent of Nbs1 status, arguing that Rif1 and Nbs1 act in different pathways to inhibit DNA replication after DNA damage. These data reveal that human Rif1 contributes to the ATM-mediated protection against DNA damage and point to a remarkable difference in the primary function of this protein in yeast and mammals (Silverman, 2004).

Direct regulation of CREB transcriptional activity by ATM in response to genotoxic stress

Ataxia-telangiectasia (A-T) is a syndrome of cancer susceptibility, immune dysfunction, and neurodegeneration that is caused by mutations in the A-T-mutated (ATM) gene. ATM has been implicated as a critical regulator of cellular responses to DNA damage, including the activation of cell cycle checkpoints and induction of apoptosis. Although defective cell cycle-checkpoint regulation and associated genomic instability presumably contribute to cancer susceptibility in A-T, the mechanism of neurodegeneration in A-T is not well understood. In addition, although ATM is required for the induction of the p53 transcriptional program in response to DNA damage, the identities of the relevant transcription factors that mediate ATM-dependent changes in gene expression remain largely undetermined. In this article, a signal transduction pathway is described linking ATM directly to the Ca(2+)/cAMP response element-binding protein, CREB, a transcription factor that regulates cell growth, homeostasis, and survival. ATM phosphorylates CREB in vitro and in vivo in response to ionizing radiation (IR) and H₂O₂ on a stress-inducible domain. IR-induced phosphorylation of CREB correlates with a decrease in CREB transactivation potential and reduced interaction between CREB and its transcriptional coactivator, CREB-binding protein (CBP). A CREB mutant containing Ala substitutions at ATM phosphorylation sites displayed enhanced transactivation potential, resistance to inhibition by IR, and increased binding to CBP. It is proposed that ATM-mediated phosphorylation of CREB in response to DNA damage modulates CREB-dependent gene expression and that dysregulation of the ATM-CREB pathway may contribute to neurodegeneration in A-T (Shi, 2004).

c-Abl and Atm in oxidative stress response are mediated by protein kinase Cdelta

c-Abl and Atm have been implicated in cell responses to DNA damage and oxidative stress. However, the molecular mechanisms by which they regulate oxidative stress response remain unclear. In this report, deficiency of c-Abl and deficiency of ATM are shown to differentially alter cell responses to oxidative stress; these signaling proteins function by induction of antioxidant protein peroxiredoxin I (Prx I) via Nrf2 and cell death, both of which require protein kinase C (PKC) delta activation and are mediated by reactive oxygen species. c-abl^-/- osteoblasts display enhanced Prx I induction, elevated Nrf2 levels, and hypersusceptibility to arsenate, which are reinstated by reconstitution of c-Abl; Atm^-/- osteoblasts show the opposite. These phenotypes correlate with increased PKC delta expression in c-abl^-/- osteoblasts and decreased PKC delta expression in Atm^-/- cells, respectively. The enhanced responses of c-abl^-/- osteoblasts can be mimicked by overexpression of PKC delta in normal cells and impeded by inhibition of PKC delta, and diminished responses of Atm^-/- cells can be rescued by PKC delta overexpression, indicating that PKC delta mediates the effects of c-Abl and ATM in oxidative stress response. Hence, these results unveiled a previously unrecognized mechanism by which c-Abl and Atm participate in oxidative stress response (Li, 2004).

How does c-Abl or Atm regulate the protein level of PKC delta? c-Abl or Atm may affect the transcription of PKC delta gene, the stability of PKC delta mRNA or protein, or the translation efficiency of PKC delta mRNA. RT-PCR assays did not reveal any significant difference in the levels of PKC delta mRNA among wild-type and c-Abl-deficient osteoblasts, suggesting that c-Abl regulates PKC delta expression posttranscriptionally. It was found that c-Abl deficiency inhibits activation-induced degradation of PKC delta, but the molecular mechanism behind this warrants further investigation. Studies have indicated that in cells expressing activated Src (Y527F), PKC delta was down-regulated. This down-regulation is a result of phosphorylation-mediated degradation. It is speculated that c-Abl, a member of the Src family, may have a similar function in regulating the level of PKC delta. It has also been shown that c-Abl interacts with PKC delta in response to oxidative stress. c-Abl is able to phosphorylate PKC delta in fibroblasts. Unfortunately, PKC delta immunoprecipitated from c-Abl-deficient and control osteoblasts did not show significant difference in phosphorylation at tyrosine residues. One possible explanation is that PKC delta might have multiple sites for tyrosine phosphorylation that are carried out by several kinases. Hence, c-Abl deficiency would not make a detectable difference. The role for Atm in the regulation of PKC delta expression is even less clear. RT-PCR analysis revealed no significant difference in the levels of PKC delta mRNA, suggesting that the regulation, like that of c-Abl, occurs at posttranscriptional levels. Surprisingly, degradation of PKC delta was similar in Atm^-/- osteoblasts and wild-type cells. One likely explanation is that the portion of degraded PKC delta molecules in Atm^-/- osteoblasts may have a shortened lifespan, whereas the rest have a normal lifespan. Treatment of Atm^-/- osteoblasts with MG132, a proteosome inhibitor, appeared to increase the PKC delta levels to that of control osteoblasts. The molecular mechanisms by which Atm regulates PKC delta protein levels need further investigation. Because Atm interacts with c-Abl and can activate it, it is possible that there exists a tertiary complex composed of PKC delta, c-Abl, and Atm in the cells, and that c-Abl may mediate the function of Atm in controlling PKC delta expression (Li, 2004).

Another layer of complexity is that Prx I/PAG is also a c-Abl interacting protein. c-Abl, a nonreceptor tyrosine kinase, plays a negative role in Prx I induction. Without c-Abl, osteoblasts show an enhanced induction of Prx I. On the basis of these facts, it is proposed that in normal osteoblasts, the induction of Prx I is suppressed, facilitating the activation of c-Abl. When c-Abl is deficient, the suppression is lifted and more Prx I is expressed. Therefore, a feedback circuit may exist that controls the activity of c-Abl in response to stress. Alternatively, interaction between c-Abl and Prx I may be involved in regulating the antioxidant activity of Prx I, for example, phosphorylation of Prx I by c-Abl. One such example is that Prx I could be phosphorylated by cdc2 and this phosphorylation reduces the activity of Prx I (Li, 2004).

Mammalian ATM and telomeres

Cells derived from ataxia telangiectasia (A-T) patients show a prominent defect at chromosome ends in the form of chromosome end-to-end associations, also known as telomeric associations, seen at G(1), G(2), and metaphase. ATM gene product, which is defective in the cancer-prone disorder A-T, influences chromosome end associations and telomere length. A possible hypothesis explaining these results is that the defective telomere metabolism in A-T cells are due to altered interactions between the telomeres and the nuclear matrix. These interactions were examined in nuclear matrix halos before and after radiation treatment. A difference was observed in the ratio of soluble versus matrix-associated telomeric DNA between cells derived from A-T and normal individuals. Ionizing radiation treatment affects the ratio of soluble versus matrix-associated telomeric DNA only in the A-T cells. To test the hypothesis that the ATM gene product is involved in interactions between telomeres and the nuclear matrix, such interactions were examined in human cells expressing either a dominant-negative effect or complementation of the ATM gene. The phenotype of RKO colorectal tumor cells expressing ATM fragments containing a leucine zipper motif mimics the altered interactions of telomere and nuclear matrix similar to that of A-T cells. A-T fibroblasts transfected with wild-type ATM gene had corrected telomere-nuclear matrix interactions. Further, A-T cells had different micrococcal nuclease digestion patterns compared to normal cells before and after irradiation, indicating differences in nucleosomal periodicity in telomeres. These results suggest that the ATM gene influences the interactions between telomeres and the nuclear matrix, and alterations in telomere chromatin could be at least partly responsible for the pleiotropic phenotypes of the ATM gene (Smilenov, 1999).

To examine the role of ataxia-telangiectasia mutated (Atm) in telomere function, Atm and telomerase null mice [Atm(-/-) mTR(-/-) iG6 mice] were generated. These mice exhibit increased germ cell death and chromosome fusions compared with either Atm(-/-) or mTR(-/-) iG6 mice. Furthermore, the Atm(-/-) mTR(--) iG6 mice have a delayed onset and reduced incidence of thymic lymphoma compared with Atm(-/-) mice. The tumors in the Atm(-/-) mTR(-/-) iG6 mice show increased apoptosis and anaphase bridges. Finally, lymphomas from Atm(-/-) mTR(-/-) iG6 mice were derived from CD8 immature, single-positive T cells, whereas Atm(-/-) lymphomas were from CD4(+)CD8(+) double-positive T cells. It is proposed that Atm protects short telomeres and that Atm deficiency cooperates with short telomeres, leading to increased cell death, decreased tumorigenesis, and increased overall survival (Qi. 2003)

Ataxia-telangiectasia results from the loss of ataxia-telangiectasia mutated (Atm) function and is characterized by accelerated telomere loss, genomic instability, progressive neurological degeneration, premature ageing and increased neoplasia incidence. The functional interaction of Atm and telomeres was examined in vivo. The impact of Atm deficiency was examined as a function of progressive telomere attrition at both the cellular and whole-organism level in mice doubly null for Atm and the telomerase RNA component (Terc). These compound mutants showed increased telomere erosion and genomic instability, yet they experienced a substantial elimination of T-cell lymphomas associated with Atm deficiency. A generalized proliferation defect was evident in all cell types and tissues examined, and this defect extended to tissue stem/progenitor cell compartments, thereby providing a basis for progressive multi-organ system compromise, accelerated ageing and premature death. Atm deficiency and telomere dysfunction act together to impair cellular and whole-organism viability, thus supporting the view that aspects of A-T pathophysiology are linked to the functional state of telomeres and its adverse effects on stem/progenitor cell reserves (Wong, 2003).

Centrosome amplification induced by DNA damage involves ATM

Centrosomes are the principal microtubule organising centers in somatic cells. Abnormal centrosome number is common in tumours and occurs after gamma-irradiation and in cells with mutations in DNA repair genes. To investigate how DNA damage causes centrosome amplification, cells were examined that conditionally lack the Rad51 recombinase and thereby incur high levels of spontaneous DNA damage. Rad51-deficient cells arrest in G2 phase and form supernumerary functional centrosomes, as assessed by light and serial section electron microscopy. This centrosome amplification occurs without an additional DNA replication round and is not the result of cytokinesis failure. G2-to-M checkpoint over-ride by caffeine or wortmannin treatment strongly reduces DNA damage-induced centrosome amplification. Radiation-induced centrosome amplification is potentiated by Rad54 disruption. Gene targeting of ATM reduces, but does not abrogate, centrosome amplification induced by DNA damage in both the Rad51 and Rad54 knockout models, demonstrating ATM-dependent and -independent components of DNA damage-inducible G2-phase centrosome amplification. These data suggest DNA damage-induced centrosome amplification as a mechanism for ensuring death of cells that evade the DNA damage or spindle assembly checkpoints (Dodson, 2004).

ATM and susceptibility to cancer

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by growth retardation, cerebellar ataxia, oculocutaneous telangiectasias, and a high incidence of lymphomas and leukemias. In addition, AT patients are sensitive to ionizing radiation. Atm-deficient mice recapitulate most of the AT phenotype. p21, an inhibitor of cyclin-dependent kinases, has been implicated in cellular senescence and response to gamma-radiation-induced DNA damage. To study the role of p21 in ATM-mediated signal transduction pathways, the combined effect of the genetic loss of atm and p21 on growth control, radiation sensitivity, and tumorigenesis were examined. p21 modifies the in vitro senescent response seen in AT fibroblasts. It is a downstream effector of ATM-mediated growth control. However, loss of p21 in the context of an atm-deficient mouse leads to a delay in thymic lymphomagenesis and an increase in acute radiation sensitivity in vivo (the latter principally because of effects on the gut epithelium). Modification of these two crucial aspects of the ATM phenotype can be related to an apparent increase in spontaneous apoptosis seen in tumor cells and in the irradiated intestinal epithelium of mice doubly null for atm and p21. Thus, loss of p21 seems to contribute to tumor suppression by a mechanism that operates via a sensitized apoptotic response. These results have implications for cancer therapy in general and AT patients in particular (Wang, 1997).

Ataxia-telangiectasia is characterized by radiosensitivity, genome instability and predisposition to cancer. Heterozygous carriers of ATM, the gene defective in ataxia-telangiectasia, have a higher than normal risk of developing breast and other cancers. Atm 'knock-in' (Atm-Delta SRI) heterozygous mice harboring an in-frame deletion corresponding to the human 7636del9 mutation show an increased susceptibility to developing tumors. In contrast, no tumors are observed in Atm knockout [Atm(+/-)] heterozygous mice. In parallel, the appearance is reported of tumors in 6 humans from 12 families who are heterozygous for the 7636del9 mutation. Expression of ATM cDNA containing the 7636del9 mutation has a dominant-negative effect in control cells, inhibiting radiation-induced ATM kinase activity in vivo and in vitro. This reduces the survival of these cells after radiation exposure and enhances the level of radiation-induced chromosomal aberrations. These results show for the first time that mouse carriers of a mutated Atm that are capable of expressing Atm have a higher risk of cancer. This finding provides further support for cancer predisposition in human ataxia-telangiectasia carriers (Spring, 2002).

ATM contributes to activation by high NaCl of the transcription factor TonEBP/OREBP

High NaCl activates the transcription factor tonicity-responsive enhancer/osmotic response element-binding protein (TonEBP/OREBP), resulting in increased transcription of several protective genes, including the glycine betaine/gamma-aminobutyric acid transporter (BGT1). High NaCl damages DNA, and DNA damage activates ataxia telangiectasia mutated (ATM) kinase through autophosphorylation on Ser-1981. TonEBP/OREBP contains ATM consensus phosphorylation sites at Ser-1197, Ser-1247, and Ser-1367. The present studies test whether ATM is involved in activation of TonEBP/OREBP by high NaCl. Raising osmolality from 300 to 500 mosmol/kg by adding NaCl activates ATM, as indicated by phosphorylation at Ser-1981. High urea and radiation also activate ATM, but they do not increase TonEBP/OREBP transcriptional activity, as does high NaCl. Wortmannin, which inhibits ATM, reduces NaCl-induced TonEBP/OREBP transcriptional activation and BGT1 mRNA increase. Overexpression of wild-type TonEBP/OREBP increases ORE/TonE reporter activity much more than does overexpression of TonEBP/OREBP S1197A, S1247A, or S1367A. In AT cells (which express nonfunctional ATM), TonEBP/OREBP transcriptional and transactivating activity are further increased by expression of wild-type ATM but not of S1981A ATM. TonEBP/OREBP reciprocally coimmunoprecipitates with ATM kinase, demonstrating physical association. Additionally, antibody to ATM kinase supershifts TonEBP/OREBP bound to its cognate ORE/TonE DNA element. In AT cells, wortmannin further decreases high NaCl-induced increase in transcriptional activity, consistent with participation of signaling kinase(s) in addition to ATM. In conclusion, signaling via ATM is necessary for full activation of TonEBP/OREBP by high NaCl, but it is not sufficient (Irarrazabal, 2004).

ATM prevents the persistence and propagation of chromosome breaks in lymphocytes

DNA double-strand breaks (DSBs) induce a signal transmitted by the ataxia-telangiectasia mutated (ATM) kinase, which suppresses illegitimate joining of DSBs and activates cell-cycle checkpoints. A significant fraction of mature ATM-deficient lymphocytes contain telomere-deleted ends produced by failed end joining during V(D)J recombination. These RAG-1/2 endonuclease-dependent, terminally deleted chromosomes persist in peripheral lymphocytes for at least 2 weeks in vivo and are stable over several generations in vitro. Restoration of ATM kinase activity in mature lymphocytes that have transiently lost ATM function leads to loss of cells with terminally deleted chromosomes. Thus, maintenance of genomic stability in lymphocytes requires faithful end joining as well a checkpoint that prevents the long-term persistence and transmission of DSBs. Silencing this checkpoint permits DNA ends produced by V(D)J recombination in a lymphoid precursor to serve as substrates for translocations with chromosomes subsequently damaged by other means in mature cells (Callén, 2007).

Duplication of Atxn1l suppresses SCA1 neuropathology by decreasing incorporation of polyglutamine-expanded ataxin-1 into native complexes

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disease caused by expansion of a glutamine tract in ataxin-1 (ATXN1). SCA1 pathogenesis studies support a model in which the expanded glutamine tract causes toxicity by modulating the normal activities of ATXN1. To explore native interactions that modify the toxicity of ATXN1, a targeted duplication of the mouse ataxin-1-like (Atxn1l, also known as Boat) locus, a highly conserved paralog of SCA1, was generated, and the role of this protein in SCA1 pathology was tested. Using a knock-in mouse model of SCA1 that recapitulates the selective neurodegeneration seen in affected individuals, it was found that elevated Atxn1l levels suppress neuropathology by displacing mutant Atxn1 from its native complex with Capicua (CIC). These results provide genetic evidence that the selective neuropathology of SCA1 arises from modulation of a core functional activity of ATXN1, and they underscore the importance of studying the paralogs of genes mutated in neurodegenerative diseases to gain insight into mechanisms of pathogenesis (Bowman, 2007).

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Search PubMed for articles about Drosophila Telomere fusion/ATM

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date revised: 2 February 2023

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