The true Drosophila ATM homolog telomere fusion (common alternative name: ATM) is more closely related to ATM, and mei-41 actually belongs to the ATR subfamily (Song, 2004).
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).
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).
The fission yeast Rad3p checkpoint protein is a member of the phosphatidylinositol 3-kinase-related family of protein kinases, which includes human ATMp. The kinase domain of Rad3p is essential for function. Although this domain is necessary, it is not sufficient, because the isolated kinase domain does not have kinase activity in vitro and cannot complement a rad3 deletion strain. Using dominant negative alleles of rad3, two sites N-terminal to the conserved kinase domain have been identified that are essential for Rad3p function. One of these sites is the putative leucine zipper, which is conserved in other phosphatidylinositol 3-kinase-related family members. The other is a novel motif, which may also mediate Rad3p protein-protein interactions (Chapman, 1999).
Genome integrity is monitored by a checkpoint that delays mitosis in response to DNA damage. This checkpoint is enforced by Chk1, a protein kinase that inhibits the mitotic inducer Cdc25. In fission yeast, Chk1 is regulated by a group of proteins that includes Rad3, a protein kinase related to human ATM and ATR. These kinases phosphorylate serine or threonine followed by glutamine (SQ/TQ). Fission yeast and human Chk1 proteins share two conserved SQ motifs at serine-345 and serine-367. Serine-345 of human Chk1 is phosphorylated in response to DNA damage. Rad3 and ATM phosphorylate serine-345 of fission yeast Chk1. Mutation of serine-345 (chk1-S345A) abrogates Rad3-dependent phosphorylation of Chk1 in vivo. The chk1-S345A cells are sensitive to DNA damage and are checkpoint defective. In contrast, mutations of serine-367 and other SQ/TQ sites do not substantially impair the checkpoint or cause damage sensitivity. These findings attest to the importance of serine-345 phosphorylation for Chk1 function and strengthen evidence that transduction of the DNA damage checkpoint signal requires direct phosphorylation of Chk1 by Rad3 (Lopez-Girona, 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 Rad3ATR occurs in S phase without replication stress. Rad3ATR and Tel1ATM 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 Rad4TOPBP1 protein. Rad9-Rad4TOPBP1 interaction is required to activate the Chk1 damage checkpoint but not the Cds1 replication checkpoint. When the Rad9-T412/S423 are phosphorylated, Rad4TOPBP1 coprecipitates with Rad3ATR, 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 Rad3ATR-dependent phosphorylation of Rad9 promotes association between Rad9 and Rad4TOPBP1 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).
Acquisition of lineage-specific cell cycle duration is a central feature of metazoan development. The mechanisms by which this is achieved during early embryogenesis are poorly understood. In the nematode Caenorhabditis elegans, differential cell cycle duration is apparent starting at the two-cell stage, when the larger anterior blastomere AB divides before the smaller posterior blastomere P1. How anterior-posterior (A-P) polarity cues control this asynchrony remains to be elucidated. Early C. elegans embryos possess a hitherto unrecognized DNA replication checkpoint that relies on the PI-3-like kinase atl-1 and the kinase chk-1. Preferential activation of this checkpoint in the P1 blastomere contributes to asynchrony of cell division in two-cell-stage wild-type embryos. Furthermore, preferential checkpoint activation is largely abrogated in embryos that undergo equal first cleavage following inactivation of Galpha signaling. These findings establish that differential checkpoint activation contributes to acquisition of distinct cell cycle duration in two-cell-stage C. elegans embryos and suggest a novel mechanism coupling asymmetric division to acquisition of distinct cell cycle duration during development (Brauchle, 2003).
This work reveals that the DNA replication checkpoint is functional in one- and two-cell-stage C. elegans embryos. Since entry into mitosis is merely delayed, even in hydroxyurea-treated embryos, this checkpoint is less potent at preventing cell cycle progression than that acting in somatic cells, where entry into mitosis is blocked following inhibition of DNA replication. Nevertheless, these findings are in contrast to observations in embryos of Drosophila, Xenopus, or Zebrafish, where aphidicolin or hydroxyurea do not affect cell cycle progression prior to the midblastula transition. Interestingly, the DNA replication checkpoint is also functional in early embryos of fucus and sea urchin; this finding indicates that the DNA replication checkpoint may contribute to modulation of cell cycle duration during early development of a number of organisms (Brauchle, 2003).
The DNA replication checkpoint in early C. elegans embryos relies on two core components of the evolutionarily conserved checkpoint signal transduction cascade: ATR-related atl-1 and chk-1. ATR is also the PI-3-like kinase seemingly required for the DNA replication checkpoint in vertebrate somatic cells, since human fibroblasts overexpressing a kinase-inactive ATR protein are hypersensitive to aphidicolin and hydroxyurea. Moreover, Xenopus egg extracts supplemented with sperm nuclei and treated with aphidicolin no longer arrest following immunodepletion of ATR. Similarly, Chk1 appears to be the main downstream effector of ATR in vertebrates. ATR phosphorylates Chk1 in Xenopus egg extracts, and this phosphorylation is essential for Chk1 to prevent entry into mitosis following DNA replication checkpoint activation. Furthermore, ATR/mei-41 and Chk1/grapes are essential for the DNA replication checkpoint in Drosophila. These observations indicate that ATR and Chk1 play a conserved role in the DNA replication checkpoint across metazoan evolution (Brauchle, 2003).
It has become increasingly clear that the DNA replication checkpoint can be utilized in the absence of defective DNA replication to modulate cell cycle progression. For instance, careful timing has revealed that ATR/mei-41 and CHK-1/grapes are required to lengthen S phases at the midblastula transition in Drosophila embryos. Moreover, immunodepletion of ATR or Chk1 from Xenopus egg extracts results in premature entry into mitosis. The current results mirror these observations and demonstrate for the first time that differential modulation of constitutive signaling can contribute to acquisition of distinct cell cycle duration during early development (Brauchle, 2003).
These findings suggest that A-P polarity regulates timing of cell division in wild-type two-cell-stage C. elegans embryos through two distinct mechanisms: (1) polarity cues govern 60% of the time difference through an atl-1/chk-1-independent mechanism that remains to be identified; (2) polarity cues result in preferential activation of the atl-1/chk-1-dependent DNA replication checkpoint in the smaller blastomere, P1, and account for the remaining 40% time difference. Why should there be two mechanisms to ensure acquisition of lineage-specific cell cycle duration? Interestingly, despite having a 40% reduction in the time difference between AB and P1, most atl-1/chk-1(RNAi) embryos give rise to viable adults, though they lack a germline. Perhaps the existence of two partially redundant mechanisms provides a means to ensure robust acquisition of distinct cell cycle duration during early embryonic development (Brauchle, 2003).
An attractive hypothesis is that preferential checkpoint activation in P1 results from the unequal first cleavage of wild-type embryos, since preferential activation is largely abrogated in embryos that undergo equal first cleavage following inactivation of Gα signaling. In apparent contradiction with this working model, par-4 mutant embryos undergo unequal first cleavage, yet exhibit near synchronous division at the two-cell stage. However, probably as a result of incomplete or delayed cytokinesis, cytoplasm often flows from AB into P1 in par-4 mutant embryos; this could alter cell cycle progression in the two blastomeres. Compatible with this view, daughter nuclei confined to a common cytoplasm following lack of cytokinesis at the one-cell stage undergo synchronous nuclear envelope breakdown. Furthermore, the apparent paradox posed by par-4 mutant embryos may be resolved if the impact of cell size on cell cycle progression can be detected in full only when polarity cues are intact. Future work, including experiments in which the first cleavage can be equalized without interfering with polarity cues or Gα signaling, will help elucidate the mechanisms by which A-P polarity ensures differential checkpoint activation during early development (Brauchle, 2003).
In screens for genetic modifiers of lin-35/Rb, the C. elegans retinoblastoma protein homolog, a mutation in xnp-1 (Drosophila homolog: XNP) was identified. Mutations in xnp-1, including a presumed null allele, are viable and, in general, appear indistinguishable from the wild type. In contrast, xnp-1 lin-35 double mutants are typically sterile and exhibit severe defects in gonadal development. Analyses of the abnormal gonads indicate a defect in the lineages that generate cells of the sheath and spermatheca. xnp-1 encodes the C. elegans homolog of ATR-X, a human disease gene associated with severe forms of mental retardation and urogenital developmental defects. xnp-1/ATR-X is a member of the Swi2/Snf2 family of ATP-dependent DEAD/DEAH box helicases, which function in nucleosome remodeling and transcriptional regulation. Expression of an xnp-1::GFP promoter fusion is detected throughout C. elegans development in several cell types including neurons and cells of the somatic gonad. These findings demonstrate a new biological role for Rb family members in somatic gonad development and implicate lin-35 in the execution of multiple cell fates in C. elegans. In addition, these results suggest a possible conserved function for xnp-1/ATR-X in gonadal development across species (Bender, 2004).
xnp-1 encodes the C. elegans homolog of the human ATR-X gene, a member of the Swi/Snf superfamily of ATP-dependent chromatin remodeling helicases. Mutations in human ATR-X lead to severe mental retardation as well as many secondary anomalies including urogenital defects in approximately 80% of ATR-X patients. The mutation identified in xnp-1(fd2) mutants leads to a substitution (R → K) of a highly conserved arginine at amino acid position 1130 (corresponding to human ATR-X position 2197) in the C terminus of the peptide. Interestingly, an analysis of molecular lesions from ATR-X patients indicates that mutations affecting the C-terminal region of the ATR-X protein are often associated with the most severe forms of urogenital defects. In contrast to humans, however, expression of the gonadal defect in C. elegans is dependent upon the coordinate inactivation of class B SynMuv genes such as lin-35. Thus, in C. elegans, lin-35 and xnp-1 function redundantly in the control of gonadal development (Bender, 2004).
Studies on Swi/Snf family members have indicated their importance in many diverse biological processes, most of which can be linked mechanistically to the control of nucleosome remodeling and gene expression. The precise level of control exerted by Swi/Snf members has been reported to range from gene-specific to global and appears to depend on several factors including the particular Swi/Snf complex involved, associations with various binding partners, genetic background, and cell cycle phase. Moreover, the effects exerted by Swi/Snf complexes on individual target genes can be either repressive or activating; the outcome most likely depends on the influence of other bound regulators such as histone modifying enzymes (Bender, 2004).
An obvious model to account for the functional redundancy of LIN-35 and XNP-1 is that both proteins share in common one or more transcriptional targets. Thus, in single-mutant backgrounds, sufficient regulation of the target can be brought about through the intact pathway acting alone. However, in double mutants, two means of regulation are missing and the shared target (or targets) may become grossly deregulated. Based on precedent from studies on the transcriptional effects of Rb family members, as well as other lin-35 synthetic mutants, the actions of both LIN-35 and XNP-1 on the shared target(s) are found to be repressive in nature (Bender, 2004).
As to how many common targets might be affected in the double mutants is an open question. Many studies analyzing the transcriptional targets of individual Swi/Snf complexes have been carried out, and they suggest that Swi/Snf proteins may regulate the expression of sizeable numbers (on the order of several hundred to several thousand) of physically disparate target genes. Likewise, many recent reports seeking to determine the transcriptional targets of Rb family members suggest that Rb family members may collectively regulate the expression of up to several hundred genes. Although such studies may be significantly compromised by issues such as cell- and tissue-type specific differences, genetic redundancy, and indirect effects, they provide at least some basis for estimating the number of genes that may be co-regulated by LIN-35 and XNP-1 in C. elegans. Namely, assuming a nonbiased set of 250 independent targets for both LIN-35 and XNP-1, as well as a genome consisting of 17,000 genes, it would be predicted that on average, 3.7 genes would be regulated by both factors. Although such calculations are highly speculative, they do suggest that the observed phenotype of xnp-1 lin-35 mutants could be due to the missexpression of a relatively small number of genes, perhaps even a single common target. Identification of such targets, either by genetics or other means, will await further studies (Bender, 2004).
Mutations in the XNP/ATR-X gene cause several X-linked mental retardation syndromes in humans. The XNP/ATR-X gene encodes a DNA-helicase belonging to the SNF2 family. It has been proposed that XNP/ATR-X might be involved in chromatin remodelling. The lack of a mouse model for the ATR-X syndrome has, however, hampered functional studies of XNP/ATR-X. C. elegans possesses one homolog of the XNP/ATR-X gene, named xnp-1. By analysing a deletion mutant, it has been shown that xnp-1 is required for the development of the embryo and the somatic gonad. Moreover, abrogation of xnp-1 function in combination with inactivation of genes of the NuRD complex, as well as lin-35/Rb and hpl-2/HP1 leads to a stereotyped block of larval development with a cessation of growth but not of cell division. A specific function for xnp-1 together with lin-35 or hpl-2 has been demonstrated in the control of transgene expression, a process known to be dependent on chromatin remodelling. This study thus demonstrates that in vivo XNP-1 acts in association with RB, HP1 and the NuRD complex during development (Cardoso, 2005).
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).
The ATR protein is a member of the phosphoinositide 3-kinase-related kinase family and plays an important role in UV-induced DNA damage checkpoint response. Its role as a signal transducer in cell cycle checkpoint is well established, but it is currently unclear whether ATR functions as a damage sensor as well. The ATR protein has been purified and its interaction with DNA was investigated by using biochemical analysis and electron microscopy. ATR has been found to be a DNA-binding protein with higher affinity to UV-damaged than undamaged DNA. In addition, damaged DNA stimulates the kinase activity of ATR to a significantly higher level than undamaged DNA. These data suggest that ATR may function as an initial sensor in the DNA damage checkpoint response (Unsal-Kacmaz, 2002).
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).
Although a small decrease in survival and increase in tumor incidence has been observed in ATR(+/-) mice, ATR(-/-) embryos die early in development, subsequent to the blastocyst stage and prior to 7.5 days p.c. In culture, ATR(-/-) blastocysts cells continue to cycle into mitosis for 2 days but subsequently fail to expand and die of caspase-dependent apoptosis. Importantly, caspase-independent chromosome breaks are observed in ATR(-/-) cells prior to widespread apoptosis, implying that apoptosis is caused by a loss of genomic integrity. These data show that ATR is essential for early embryonic development and must function in processes other than regulation of p53 (Brown, 2000).
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).
A Cre/lox-conditional mouse line was generated to evaluate the role of ATR in checkpoint responses to ionizing radiation (IR) and stalled DNA replication. After IR treatment, ATR and ATM each contribute to early delay in M-phase entry but ATR regulates a majority of the late phase (2-9 h post-IR). Double deletion of ATR and ATM eliminates nearly all IR-induced delay, indicating that ATR and ATM cooperate in the IR-induced G2/M-phase checkpoint. In contrast to the IR-induced checkpoint, checkpoint delay in response to stalled DNA replication is intact in ATR knockout cells and ATR/ATM and ATR/p53 double-knockout cells. The DNA replication checkpoint remains intact in ATR knockout cells even though the checkpoint-stimulated inhibitory phosphorylation of Cdc2 on T14/Y15 and activating phosphorylation of the Chk1 kinase no longer occur. Thus, incomplete DNA replication in mammalian cells can prevent M-phase entry independently of ATR and inhibitory phosphorylation of Cdc2. When DNA replication inhibitors are removed, ATR knockout cells proceed to mitosis but do so with chromosome breaks, indicating that ATR provides a key genome maintenance function in S phase (Brown, 2003).
The human homologue of the C. elegans biological clock protein CLK-2 (HCLK2) associates with the S-phase checkpoint components ATR, ATRIP, claspin and Chk1. Consistent with a critical role in the S-phase checkpoint, HCLK2-depleted cells accumulate spontaneous DNA damage in S-phase, exhibit radio-resistant DNA synthesis, are impaired for damage-induced monoubiquitination of FANCD2 and fail to recruit FANCD2 and Rad51 (critical components of the Fanconi anaemia and homologous recombination pathways, respectively) to sites of replication stress. Although Thr 68 phosphorylation of the checkpoint effector kinase Chk2 remains intact in the absence of HCLK2, claspin phosphorylation and degradation of the checkpoint phosphatase Cdc25A are compromised following replication stress as a result of accelerated Chk1 degradation. ATR phosphorylation is known to both activate Chk1 and target it for proteolytic degradation, and depleting ATR or mutation of Chk1 at Ser 345 restored Chk1 protein levels in HCLK2-depleted cells. It is concluded that HCLK2 promotes activation of the S-phase checkpoint and downstream repair responses by preventing unscheduled Chk1 degradation by the proteasome (Collis, 2007).
Although the link between transcription and DNA repair is well established, defects in the core transcriptional complex itself have not been shown to elicit a DNA damage response. A cell line with a temperature-sensitive defect in TBP-associated factor 1 (TAF1), a component of the TFIID general transcription complex, exhibits hallmarks of an ATR-mediated DNA damage response. Upon inactivation of TAF1, ATR rapidly localized to subnuclear foci and contributed to the phosphorylation of several downstream targets, including p53 and Chk1, resulting in cell cycle arrest. The increase in p53 expression and the G(1) phase arrest can be blocked by caffeine, an inhibitor of ATR. In addition, dominant negative forms of ATR but not ATM are able to override the arrest in G(1). These results suggest that a defect in TAF1 can elicit a DNA damage response (Buchmann, 2004).
Conditions that partially inhibit DNA replication induce expression of common fragile sites. These sites form gaps and breaks on metaphase chromosomes and are deleted and rearranged in many tumors. Yet an understanding of the mechanism of fragile site expression has remained elusive. The replication checkpoint kinase ATR, but not ATM, is critical for maintenance of fragile site stability. ATR deficiency results in fragile site expression with and without addition of replication inhibitors. Thus, it is proposed that fragile sites are unreplicated chromosomal regions resulting from stalled forks that escape the ATR replication checkpoint. These findings have important implications for understanding both the mechanism of fragile site instability and the consequences of stalled replication in mammalian cells (Casper, 2002).
Common fragile sites are loci that exhibit gaps and breaks on metaphase chromosomes of cells that have been cultured under conditions of replicative stress, such as folate deficiency or treatment with aphidicolin. Unlike rare fragile sites, which result from expanded di- or tri-nucleotide repeat mutation, common fragile sites do not contain such repeats and are a normal component of chromosome structure. The exact number of common fragile sites that exist is a matter of interpretation, and 75 aphidicolin-induced common fragile sites are listed in Genbank. Increasing the stress placed on DNA replication leads to the expression of increasing numbers of fragile sites, until replication stops altogether. However, gaps and breaks at just 20 fragile sites represent over 80% of all lesions observed in lymphocytes following treatment with low doses of aphidicolin. FRA3B at 3p14.2 stands out as the most 'fragile' site in the genome, followed by 16q23 (FRA16D), 6q26 (FRA6E), 7q31.2 (FRA7G), and Xp22.3 (FRAXB) (Casper, 2002).
Common fragile sites are normally stable in cultured human cells. However, following induction with replication inhibitors, these sites are 'hot spots' for increased sister chromatid exchanges (SCE), translocations, and deletions. They are preferred sites of plasmid integration and may be favored targets for DNA virus integration in vivo. Fragile sites may also play a role in some gene amplification events, both in vitro and in tumor cells, by triggering a breakage-fusion-bridge cycle. All of these chromosomal alterations are preceded by a DNA double-strand (DS) break, leading to the conclusion that DS breaks are sometimes associated with fragile site expression (Casper, 2002).
Five fragile sites have been cloned and characterized and have been found to extend over hundreds of kilobases, with gaps or breaks on metaphase chromosomes occurring throughout these regions. Numerous studies have shown that these sites are unstable in tumor cells. For example, FHIT, the gene spanning FRA3B, is often rearranged or partially deleted in many tumors, including lung, breast, ovarian, cervical, and esophageal. Investigation of chromosome 3 homologs from tumor cell lines shows multiple variable deletions within FRA3B, suggesting ongoing instability in the region. WWOX, the gene at FRA16D, shows loss of heterozygosity and deletions in various cancers, as do FRA7G, FRA7H, and FRAXB. Through functional studies, both FHIT and WWOX have been identified as tumor suppressors. However, the genes identified at FRAXB are not involved in tumor progression, indicating that fragile site instability in tumors is not driven solely by associated gene function. All of these results support the hypothesis that fragile sites are involved in chromosome rearrangements in cancer (Casper, 2002).
Determining the mechanism of common fragile site expression is important in understanding a normal component of chromosome structure and function and in understanding the instability found at fragile sites in tumor cells. Beyond the knowledge that partial inhibition of DNA synthesis is involved, little is known about the mechanism of common fragile site expression. Sequence analyses have not readily revealed why the sites are unstable; however, all fragile sites studied to date are relatively AT-rich. Studies of replication timing at FRA3B have found this site to be late replicating, as had previously been shown for the fragile X site (FRAXA). Following addition of aphidicolin, FRA3B replicates even later, and in some cells may remain unreplicated in G2. Replication timing at FRA7H has also been studied and a detailed analysis indicates significant differences in the replication timing of adjacent segments with some segments replicating late in S phase, a pattern that was exaggerated by the addition of aphidicolin. These results suggest that late replication may play a role in fragile site expression (Casper, 2002).
Based on the occurrence of DNA breaks and chromosome rearrangements at fragile site loci and the possible role of replication fork stalling at these sites, it was hypothesized that the S phase and G2/M cellular checkpoint proteins ATM and ATR are involved in fragile site maintenance and stability. Recent findings show that ATM and ATR act in distinct but partially overlapping pathways in response to specific types of DNA damage during cellular replication. ATM has been studied in cell lines derived from AT patients and is activated by DNA DS breaks. Thus, AT cells are sensitive to ionizing irradiation but not UV-light. ATR has been more difficult to study, because knockout mice die in early embryogenesis, and cells lacking this protein are inviable within a few cell divisions. ATR has been shown to be required for checkpoint responses after treatment of cells with UV light and agents that block replication fork progression, such as hydroxyurea and aphidicolin. A major outcome of ATR deficiency found with these approaches was extreme chromosome fragmentation following treatment of cells with high concentrations of aphidicolin or hydroxyurea. The current model is that ATM and ATR perform critical early functions to activate the replication checkpoints in response to DNA DS breaks or stalled replication forks, respectively (Casper, 2002).
The role of ATM and ATR in the maintenance of fragile site stability has been investigated. An inhibitor of ATM and ATR kinases, 2-aminopurine (2-AP), increases fragile site expression. AT cell lines were used to study ATM and three methods were used to study ATR: (1) a dominant-negative approach in stably transfected cell lines; (2) cre-lox mediated ATR deficiency in cell lines with lox P-flanked ATR, and (3) RNAi using siRNA duplexes directed against ATR. Fragile site expression was found to be unchanged in AT cells, indicating that ATM is not required for the maintenance of fragile site stability. In contrast, partial inhibition of ATR causes a 5- to 20-fold increase in aphidicolin-induced fragile site expression compared to control cells. Furthermore, fragile sites are expressed in ATR-deficient cells without addition of replication inhibitors. These results demonstrate that the ATR checkpoint pathway is critical for the maintenance of stability at common fragile sites. This finding is an important advance in understanding the mechanism of fragile site maintenance in normal cells and chromosome rearrangements at fragile sites in tumor cells (Casper, 2002).
Nijmegen breakage syndrome (NBS) is characterised by microcephaly, developmental delay, characteristic facial features, immunodeficiency and radiosensitivity. Nbs1 (Drosophila homolog Nbs)), the protein defective in NBS, functions in ataxia telangiectasia mutated protein (ATM)-dependent signalling likely facilitating ATM phosphorylation events. While NBS shares overlapping characteristics with ataxia telangiectasia, it also has features overlapping with ATR-Seckel (ATR: ataxia-telangiectasia and Rad3-related protein) syndrome, a subclass of Seckel syndrome mutated in ATR. Nbs1 also facilitates ATR-dependent phosphorylation. NBS cell lines show a similar defect in ATR phosphorylation of Chk1, c-jun and p-53 in response to UV irradiation- and hydroxyurea (HU)-induced replication stalling. They are also impaired in ubiquitination of FANCD2 after HU treatment, which is ATR dependent. Following HU-induced replication arrest, NBS and ATR-Seckel cells show similarly impaired G2/M checkpoint arrest and an impaired ability to restart DNA synthesis at stalled replication forks. Moreover, NBS cells fail to retain ATR in the nucleus following HU treatment and extraction. These findings suggest that Nbs1 functions in both ATR- and ATM-dependent signalling. It is proposed that the NBS clinical features represent the result of these combined defects (Stiff, 2005).
The ATR-dependent DNA damage response pathway can respond to a diverse group of lesions as well as inhibitors of DNA replication. Using the Xenopus egg extract system, it has been shown that lesions induced by UV irradiation and cis-platinum cause the functional uncoupling of MCM helicase and DNA polymerase activities, an event previously shown for aphidicolin. Inhibition of uncoupling during elongation with inhibitors of MCM7 or Cdc45, a putative helicase cofactor, results in abrogation of Chk1 phosphorylation, indicating that uncoupling is necessary for activation of the checkpoint. However, uncoupling is not sufficient for checkpoint activation, and DNA synthesis by Polalpha is also required. Finally, using plasmids of varying size, it has been demonstrated that all of the unwound DNA generated at a stalled replication fork can contribute to the level of Chk1 phosphorylation, suggesting that uncoupling amplifies checkpoint signaling at each individual replication fork. Taken together, these observations indicate that functional uncoupling of MCM helicase and DNA polymerase activities occurs in response to multiple forms of DNA damage and that there is a general mechanism for generation of the checkpoint-activating signal following DNA damage (Byun, 2005).
One critical component of the DNA damage response pathway is the ATR-ATRIP complex. ATR is a phosphatidylinositol kinase-related protein kinase that is thought to function as both a sensor and transducer in the DNA damage response. ATR, and its associated protein ATRIP, respond to a broad spectrum of genotoxic agents that includes ultraviolet light, topoisomerase inhibitors, alkylating agents, and cis-platinum, as well as chemicals that disrupt replication, such as aphidicolin and hydroxyurea (HU). Following DNA damage, ATR phosphorylates and activates the checkpoint kinase Chk1. In higher eukaryotes, the phosphorylation of Chk1 also requires the activities of the Rad9-Rad1-Hus1 (RHR, aka 9-1-1) complex and Claspin. The RHR complex is a PCNA-related complex that is loaded on to primed DNA in vitro and is recruited to sites of DNA damage in vivo. Claspin was initially identified as a protein that binds the activated form of Chk1, and it has been shown to bind chromatin throughout S phase (Byun, 2005 and references therein).
Recruitment of ATR and Rad1 to chromatin and activation of Chk1 requires DNA replication in Xenopus egg extracts following several types of DNA damage. Studies in mammalian cells also indicate that ATR binds UV-damaged chromatin in S phase but not G1 phase. In addition, other studies show that a replication fork must be established in Saccharomyces cerevisiae for checkpoint activation induced by methylmethane sulfonate (MMS). Taken together, these observations suggest that one or more replication-dependent events are needed to generate the signal that ATR recognizes for many types of DNA damage (Byun, 2005 and references therein).
Although the exact nature of the biochemical signal(s) responsible for activating the ATR pathway and the replication-dependent steps necessary for its formation are still unclear, evidence from a number of different systems supports a central role for replication protein A (RPA)-coated single-stranded DNA (ssDNA) in the response. In yeast, certain RPA mutants exhibit a checkpoint defect and also adapt more rapidly to DNA damage. In addition, knock-down of the ssDNA-binding protein RPA results in a significant loss of both Chk1 phosphorylation and ATR foci formation following DNA damage in mammalian cells. In Xenopus egg extracts, RPA is also required for the recruitment of ATR to chromatin following treatment with aphidicolin or etoposide and for the recruitment of ATR to poly(dA)70 ssDNA. Importantly, in vitro experiments have shown that RPA is sufficient for the binding of ATRIP to ssDNA and that RPA also facilitates the association of the RHR complex with DNA (Byun, 2005 and references therein).
Interestingly, the amount of ssDNA appears to increase following genotoxic stress, as RPA accumulates on chromatin in Xenopus extracts and mammalian cells treated with UV, MMS, HU, or aphidicolin. Moreover, in budding yeast, increased amounts of ssDNA have been observed by electron microscopy following HU treatment. In the case of DNA damage, the mechanism by which this ssDNA accumulates is not known, nor is it clear if ssDNA accumulation is required for checkpoint activation. In principle, a number of DNA repair (e.g., nucleotide excision repair, base excision repair) and recombination processes could lead to the generation of ssDNA following DNA damage at several points in the cell cycle. Alternatively, during DNA replication, ssDNA could be formed if DNA polymerases are slowed by lesions and the replicative helicase continues to unwind DNA. Indeed, uncoupling of helicase and polymerase activities has been observed in the presence of aphidicolin, and recent studies have shown that this aphidicolin-induced uncoupling is dependent on the MCM helicase (Byun, 2005 and references therein).
This uses a cell-free extract system derived from Xenopus eggs to examine the mechanism by which ssDNA accumulates following DNA damage. The appearance and disappearance of a highly unwound form of plasmid DNA that accumulates following aphidicolin treatment correlates with the phosphorylation of Chk1 on Ser 344 (S344). Importantly, this hyperunwound form of DNA is also observed upon replication of plasmid DNA damaged with either UV or cis-platinum. This suggests that DNA damage induces uncoupling of helicase and polymerase activities and that these lesions, as well as aphidicolin, may generate a common checkpoint-activating DNA structure. Moreover, while stalling the replication fork with aphidicolin results in a robust checkpoint response, it is found that aphidicolin elicits no checkpoint when the MCM DNA helicase is inactivated. Using plasmids of varying sizes, it is also shown that functional uncoupling of DNA unwinding and DNA synthesis during S phase may serve to amplify the level of Chk1 phosphorylation that can be achieved at each individual replication fork. Finally, it is demonstrated that although DNA unwinding is necessary for checkpoint activation, it is not sufficient and additional DNA synthesis by Pol is needed. Taken together, these results suggest that functional uncoupling of helicase and polymerase activities is necessary to convert DNA lesions and chemical inhibitors of DNA replication into the signal(s) that activate the ATR-dependent checkpoint (Byun, 2005).
In response to DNA damage and replication blocks, cells prevent cell cycle progression through the control of critical cell cycle regulators. This study reports the identification of Chk2, the mammalian homolog of the Saccharomyces cerevisiae Rad53 and Schizosaccharomyces pombe Cds1 protein kinases required for the DNA damage and replication checkpoints. Chk2 is rapidly phosphorylated and activated in response to replication blocks and DNA damage; the response to DNA damage occurs in an ATM-dependent manner. In vitro, Chk2 phosphorylates Cdc25C on serine-216, a site known to be involved in negative regulation of Cdc25C. This is the same site phosphorylated by the protein kinase Chk1, which suggests that, in response to DNA damage and DNA replicational stress, Chk1 and Chk2 may phosphorylate Cdc25C to prevent entry into mitosis (Matsuoka, 1998).
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).
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).
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).
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).
Chk1 is an evolutionarily conserved protein kinase that regulates cell cycle progression in response to checkpoint activation. Agents that block DNA replication or cause certain forms of DNA damage induce the phosphorylation of human Chk1. The phosphorylated form of Chk1 possesses higher intrinsic protein kinase activity and elutes more quickly on gel filtration columns. Serines 317 and 345 were identified as sites of phosphorylation in vivo, and ATR (the ATM- and Rad3-related protein kinase) phosphorylates both of these sites in vitro. Furthermore, phosphorylation of Chk1 on serines 317 and 345 in vivo is ATR dependent. Mutants of Chk1 containing alanine in place of serines 317 and 345 are poorly activated in response to replication blocks or genotoxic stress in vivo, are poorly phosphorylated by ATR in vitro, and are not found in faster-eluting fractions by gel filtration. These findings demonstrate that the activation of Chk1 in response to replication blocks and certain forms of genotoxic stress involves phosphorylation of serines 317 and 345. In addition, this study implicates ATR as a direct upstream activator of Chk1 in human cells (Zhao, 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).
The tumor suppressor gene CHK2 encodes a versatile effector serine/threonine kinase involved in responses to DNA damage. Chk2 has an amino-terminal SQ/TQ cluster domain (SCD), followed by a forkhead-associated (FHA) domain and a carboxyl-terminal kinase catalytic domain. Mutations in the SCD or FHA domain impair Chk2 checkpoint function. Autophosphorylation of Chk2 produced in a cell-free system requires trans phosphorylation by a wortmannin-sensitive kinase, probably ATM or ATR. Both SQ/TQ sites and non-SQ/TQ sites within the Chk2 SCD can be phosphorylated by active Chk2. Amino acid substitutions in the SCD and the FHA domain impair auto- and trans-kinase activities of Chk2. Chk2 forms oligomers that minimally require the FHA domain of one Chk2 molecule and the SCD within another Chk2 molecule. Chk2 oligomerization in vivo increases after DNA damage, and when damage is induced by gamma irradiation, this increase requires ATM. Chk2 oligomerization is phosphorylation dependent and can occur in the absence of other eukaryotic proteins. Chk2 can cross-phosphorylate another Chk2 molecule in an oligomeric complex. Induced oligomerization of a Chk2 chimera in vivo, concomitant with limited DNA damage augments Chk2 kinase activity. These results suggest that Chk2 oligomerization regulates Chk2 activation, signal amplification, and transduction in DNA damage checkpoint pathways (Xu, 2002).
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).
The promyelocytic leukaemia (PML) gene is translocated in most acute promyelocytic leukaemias and encodes a tumor suppressor protein. PML is involved in multiple apoptotic pathways and is thought to be pivotal in gamma irradiation-induced apoptosis. The DNA damage checkpoint kinase hCds1/Chk2 is necessary for p53-dependent apoptosis after gamma irradiation. In addition, gamma irradiation-induced apoptosis also occurs through p53-independent mechanisms, although the molecular mechanism remains largely unknown. hCds1/Chk2 is shown to mediate gamma irradiation-induced apoptosis in a p53-independent manner through an ataxia telangiectasia-mutated (ATM)-hCds1/Chk2-PML pathway. These results provide the first evidence of a functional relationship between PML and a checkpoint kinase in gamma irradiation-induced apoptosis (Yang, 2002).
Inhibition of replicon initiation is a stereotypic DNA damage response mediated through S checkpoint mechanisms not yet fully understood. Studies were undertaken to elucidate the function of checkpoint proteins in the inhibition of replicon initiation following irradiation with 254 nm UV light (UVC) of diploid human fibroblasts immortalized by the ectopic expression of telomerase. Velocity sedimentation analysis of nascent DNA molecules has revealed a 50% inhibition of replicon initiation when normal human fibroblasts were treated with a low dose of UVC. Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and AT-like disorder fibroblasts, which lack an S checkpoint response when exposed to ionizing radiation, responded normally when exposed to UVC and inhibited replicon initiation. Pretreatment of normal and AT fibroblasts with caffeine or UCN-01, inhibitors of ATR (AT mutated and Rad3 related) and Chk1, respectively, abolish the S checkpoint response to UVC. Moreover, overexpression of kinase-inactive ATR in U2OS cells severely attenuates UVC-induced Chk1 phosphorylation and reverses the UVC-induced inhibition of replicon initiation, as does overexpression of kinase-inactive Chk1. Taken together, these data suggest that the UVC-induced S checkpoint response of inhibition of replicon initiation is mediated by ATR signaling through Chk-1 and is independent of ATM, Nbs1, and Mre11 (Heffernan, 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).
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).
The checkpoint mediator protein Claspin is essential for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing aphidicolin-induced DNA replication blocks. During this checkpoint response, Claspin becomes phosphorylated on threonine 906 (T906), which creates a docking site for Plx1, the Xenopus Polo-like kinase. This interaction promotes the phosphorylation of Claspin on a nearby serine (S934) by Plx1. After a prolonged interphase arrest, aphidicolin-treated egg extracts typically undergo adaptation and enter into mitosis despite the presence of incompletely replicated DNA. In this process, Claspin dissociates from chromatin, and Chk1 undergoes inactivation. By contrast, aphidicolin-treated extracts containing mutants of Claspin with alanine substitutions at positions 906 or 934 (T906A or S934A) are unable to undergo adaptation. Under such adaptation-defective conditions, Claspin accumulates on chromatin at high levels, and Chk1 does not decrease in activity. These results indicate that the Plx1-dependent inactivation of Claspin results in termination of a DNA replication checkpoint response (Yoo, 2004).
Claspin (potential Drosophila homolog: CG32251) is required for the phosphorylation and activation of the Chk1 protein kinase by ATR during DNA replication and in response to DNA damage. This checkpoint pathway plays a critical role in the resistance of cells to genotoxic stress. Human Claspin is cleaved by caspase-7 during the initiation of apoptosis. In cells, induction of DNA damage by etoposide at first produced rapid phosphorylation of Chk1 at a site targeted by ATR. Subsequently, etoposide causes activation of caspase-7, cleavage of Claspin, and dephosphorylation of Chk1. In apoptotic cell extracts, Claspin is cleaved by caspase-7 at a single aspartate residue into a large N-terminal fragment and a smaller C-terminal fragment that each contain different functional domains. The large N-terminal fragment was heavily phosphorylated in a human cell-free system in response to double-stranded DNA oligonucleotides, and this fragment retained Chk1 binding activity. In contrast, the smaller C-terminal fragment did not bind Chk1, but did associate with DNA and inhibited the DNA-dependent phosphorylation of Chk1 associated with its activation. These results indicate that cleavage of Claspin by caspase-7 inactivates the Chk1 signaling pathway. This mechanism may regulate the balance between cell cycle arrest and induction of apoptosis during the response to genotoxic stress (Clarke, 2005).
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 ARF tumour suppressor (p14ARF in humans, p19ARF in mice) is a central component of the cellular defence against oncogene activation. The expression of ARF, which shares a genetic locus with the p16INK4a tumour suppressor, is regulated by the action of transcription factors such as members of the E2F family. ARF can bind to and inhibit the Hdm2 protein (Mdm2 in mice), which functions as an inhibitor and E3 ubiquitin ligase for the p53 transcription factor. In addition to activating p53 through binding Mdm2, ARF possesses other functions, including an ability to repress the transcriptional activity of the antiapoptotic RelA(p65) NF-kappaB subunit. ARF induces the ATR- and Chk1-dependent phosphorylation of the RelA transactivation domain at threonine 505, a site required for ARF-dependent repression of RelA transcriptional activity. Consistent with this effect, ATR and Chk1 are required for ARF-induced sensitivity to tumour necrosis factor-alpha induced cell death. Significantly, ATR activity is also required for ARF-induced p53 activity and inhibition of proliferation. ARF achieves these effects by activating ATR and Chk1. Furthermore, ATR and its scaffold protein BRCA1, but not Chk1, relocalise to specific nucleolar sites. These results reveal novel functions for ARF, ATR and Chk1 together with a new pathway regulating RelA NF-kappaB function. Moreover, this pathway provides a mechanism through which ARF can remodel the cellular response to an oncogenic challenge and execute its function as a tumour suppressor (Rocha, 2005).
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).
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).
The checkpoint kinases ATM and ATR transduce genomic stress signals to halt cell cycle progression and promote DNA repair. This study reports the identification of an ATR-interacting protein (ATRIP) that is phosphorylated by ATR, regulates ATR expression, and is an essential component of the DNA damage checkpoint pathway. ATR and ATRIP both localize to intranuclear foci after DNA damage or inhibition of replication. Deletion of ATR mediated by the Cre recombinase causes the loss of ATR and ATRIP expression, loss of DNA damage checkpoint responses, and cell death. Therefore, ATR is essential for the viability of human somatic cells. Small interfering RNA directed against ATRIP causes the loss of both ATRIP and ATR expression and the loss of checkpoint responses to DNA damage. Thus, ATRIP and ATR are mutually dependent partners in cell cycle checkpoint signaling pathways (Cortez, 2001).
The function of the ATR-ATRIP protein kinase complex is crucial for the cellular response to replication stress and DNA damage. Replication protein A (RPA), a protein complex that associates with single-stranded DNA (ssDNA), is required for the recruitment of ATR to sites of DNA damage and for ATR-mediated Chk1 activation in human cells. In vitro, RPA stimulates the binding of ATRIP to ssDNA. The binding of ATRIP to RPA-coated ssDNA enables the ATR-ATRIP complex to associate with DNA and stimulates phosphorylation of the Rad17 protein that is bound to DNA. Furthermore, Ddc2, the budding yeast homolog of ATRIP, is specifically recruited to double-strand DNA breaks in an RPA-dependent manner. A checkpoint-deficient mutant of RPA, rfa1-t11, is defective for recruiting Ddc2 to ssDNA both in vivo and in vitro. These data suggest that RPA-coated ssDNA is the critical structure at sites of DNA damage that recruits the ATR-ATRIP complex and facilitates its recognition of substrates for phosphorylation and the initiation of checkpoint signaling (Zou, 2003).
Genotoxic stress triggers the activation of checkpoints that delay cell-cycle progression to allow for DNA repair. Studies in fission yeast implicate members of the Rad family of checkpoint proteins: these include Rad17, Rad1, Rad9 and Hus1 as key early-response elements during the activation of both the DNA damage and replication checkpoints. A direct regulatory linkage between the human Rad17 homolog (hRad17) and the checkpoint kinases, ATM and ATR, has been demonstrated. Treatment of human cells with genotoxic agents induces ATM/ATR-dependent phosphorylation of hRad17 at Ser 635 and Ser 645. Overexpression of a hRad17 mutant (hRad17AA) bearing Ala substitutions at both phosphorylation sites abrogates the DNA-damage-induced G2 checkpoint, and sensitizes human fibroblasts to genotoxic stress. In contrast to wild-type hRad17, the hRad17AA mutant shows no ionizing-radiation-inducible association with hRad1, a component of the hRad1-hRad9-hHus1 checkpoint complex. These findings demonstrate that ATR/ATM-dependent phosphorylation of hRad17 is a critical early event during checkpoint signalling in DNA-damaged cells (Bao, 2001).
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).
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).
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).
An analysis was performed of how single-strand DNA gaps affect DNA replication in Xenopus egg extracts. DNA lesions generated by etoposide, a DNA topoisomerase II inhibitor, or by exonuclease treatment activate a DNA damage checkpoint that blocks initiation of plasmid and chromosomal DNA replication. The checkpoint is abrogated by caffeine and requires ATR, but not ATM, protein kinase. The block to DNA synthesis is due to inhibition of Cdc7/Dbf4 protein kinase activity and the subsequent failure of Cdc45 to bind to chromatin. The checkpoint does not require pre-RC assembly but requires loading of the single-strand binding protein, RPA, on chromatin. This is the biochemical demonstration of a DNA damage checkpoint that targets Cdc7/Dbf4 protein kinase (Costanzo, 2003).
Upon damage of DNA in eukaryotic cells, several repair and checkpoint proteins undergo a dramatic intranuclear relocalization, translocating to nuclear foci thought to represent sites of DNA damage and repair. Examples of such proteins include the checkpoint kinase ATR (ATM and Rad3-related) as well as replication protein A (RPA), a single-stranded DNA binding protein required in DNA replication and repair. A microscopy-based approach has been used to investigate whether the damage-induced translocation of RPA is an active process regulated by ATR. In undamaged cells, ATR and RPA are uniformly distributed in the nucleus or localized to promyelocytic leukemia protein (PML) nuclear bodies. In cells treated with ionizing radiation, both ATR and RPA translocate to punctate, abundant nuclear foci where they continue to colocalize. Surprisingly, an ATR mutant that lacks kinase activity fails to relocalize in response to DNA damage. Furthermore, this kinase-inactive mutant blocks the translocation of RPA in a cell cycle-dependent manner. These observations demonstrate that the kinase activity of ATR is essential for the irradiation-induced release of ATR and RPA from PML bodies and translocation of ATR and RPA to potential sites of DNA damage (Barr, 2003).
How might ATR regulate the localization of RPA? RPA has been shown to be a substrate for ATR, raising the possibility that ATR mediates the translocation of RPA through direct phosphorylation. However, mutation of two consensus ATR phosphorylation sites in RPA does not affect the ability of RPA to form ionizing radiation-induced nuclear foci. Although it is still possible that phosphorylation of an unidentified ATR-specific site in RPA is involved, it seems more likely that ATR's effects on RPA localization may be indirect (Barr, 2003).
Using the Xenopus egg extract system, the involvement of DNA replication in activation of the DNA damage checkpoint was investigated. DNA damage slows replication in a checkpoint-independent manner and is accompanied by replication-dependent recruitment of ATR and Rad1 to chromatin. The replication proteins RPA and Polalpha accumulate on chromatin following DNA damage. Finally, damage-induced Chk1 phosphorylation and checkpoint arrest are abrogated when replication is inhibited. These data indicate that replication is required for activation of the DNA damage checkpoint and suggest a unifying model for ATR activation by diverse lesions during S phase (Lupardus, 2002).
Cyclin E, in conjunction with its catalytic partner cdk2, is rate limiting for entry into the S phase of the cell cycle. Cancer cells frequently contain mutations within the cyclin D-Retinoblastoma protein pathway that lead to inappropriate cyclin E-cdk2 activation. Although deregulated cyclin E-cdk2 activity is believed to directly contribute to the neoplastic progression of these cancers, the mechanism of cyclin E-induced neoplasia is unknown. The consequences of deregulated cyclin E expression have been studied in primary cells; cyclin E was found to initiate a p53-dependent response that prevents excess cdk2 activity by inducing expression of the p21Cip1 cdk inhibitor. The increased p53 activity is not associated with increased expression of the p14ARF tumor suppressor. Instead, cyclin E leads to increased p53 serine15 phosphorylation that is sensitive to inhibitors of the ATM/ATR family. When either p53 or p21cip1 is rendered nonfunctional, then the excess cyclin E becomes catalytically active and causes defects in S phase progression, increased ploidy, and genetic instability. It is concluded that p53 and p21 form an inducible barrier that protects cells against the deleterious consequences of cyclin E-cdk2 deregulation. A response that restrains cyclin E deregulation is likely to be a general protective mechanism against neoplastic transformation. Loss of this response may thus be required before deregulated cyclin E can become fully oncogenic in cancer cells. Furthermore, the combination of excess cyclin E and p53 loss may be particularly genotoxic, because cells cannot appropriately respond to the cell cycle anomalies caused by excess cyclin E-cdk2 activity (Minella, 2002).
How might deregulated cyclin E cause S phase abnormalities that activate an S phase checkpoint? In yeast, S phase-promoting cyclins inhibit the transition of replication origins to the prereplicative state. Furthermore, when early-firing origins are inhibited by hydroxyurea, then the stalled early origins inhibit late origins through a checkpoint that requires the Mec1 protein (the budding yeast ATM/ATR homolog. Similarly, inhibition of ATR function in a human cell line by a kinase-inactive ATR mutant renders these cells hypersensitive to treatments that prolong DNA synthesis, and cyclin E overexpression is synthetically lethal with ATR inhibition. Thus, perhaps cyclin E deregulation leads to aberrant licensing of replication origins, and the resultant S phase progression defect may be sensed by a protein such as ATR, which then enforces an S phase checkpoint. Furthermore, the stalled replication origins associated with this prolonged S phase may be fragile and constitute the precursors to genetic instability. Another mechanism through which enforced cyclin E expression might impair normal cell cycle progression is by cyclin A-cdk2 inhibition, since cyclin A-cdk2 activity (and cyclin A expression) drops substantially in cells with ectopic cyclin E expression. However, cyclin E-induced cell cycle anomalies persist in E6-expressing cells with high levels of cyclin A-cdk2 kinase activity, so cyclin A-cdk2 activity cannot be the principle cause of the cyclin E-associated S phase phenotype (Minella, 2002).
Melanomas accumulate a high burden of mutations that could potentially generate neoantigens, yet somehow suppress the immune response to facilitate continued growth. This study identified a subset of human melanomas that have loss-of-function mutations in ATR (see Drosophila Meiotic 41), a kinase that recognizes and repairs UV-induced DNA damage and is required for cellular proliferation. ATR mutant tumors exhibit both the accumulation of multiple mutations and the altered expression of inflammatory genes, resulting in decreased T cell recruitment and increased recruitment of macrophages known to spur tumor invasion. Taken together, these studies identify a mechanism by which melanoma cells modulate the immune microenvironment to promote continued growth (Chen, 2017).
Microcephaly and medulloblastoma may both result from mutations that compromise genomic stability. This study reports that ATR (see Drosophila ATR), which is mutated in the microcephalic disorder Seckel syndrome, sustains cerebellar growth by maintaining chromosomal integrity during postnatal neurogenesis. Atr deletion in cerebellar granule neuron progenitors (CGNPs) induced proliferation-associated DNA damage, p53 activation (see Drosophila p53), apoptosis and cerebellar hypoplasia in mice. Co-deletions of either p53 or Bax and Bak prevented apoptosis in Atr-deleted CGNPs, but failed to fully rescue cerebellar growth. ATR-deficient CGNPs had impaired cell cycle checkpoint function and continued to proliferate, accumulating chromosomal abnormalities. RNA-Seq demonstrated that the transcriptional response to ATR-deficient proliferation was highly p53 dependent and markedly attenuated by p53 co-deletion. Acute ATR inhibition in vivo by nanoparticle-formulated VE-822 reproduced the developmental disruptions seen with Atr deletion. Genetic deletion of Atr blocked tumorigenesis in medulloblastoma-prone SmoM2 mice. These data show that p53-driven apoptosis and cell cycle arrest - and, in the absence of p53, non-apoptotic cell death - redundantly limit growth in ATR-deficient progenitors. These mechanisms may be exploited for treatment of CGNP-derived medulloblastoma using ATR inhibition (Lang, 2016).
Fanconi anemia (FA) is a multigenic autosomal recessive cancer susceptibility syndrome. The FA pathway regulates the monoubiquitination of FANCD2 and the assembly of damage-associated FANCD2 nuclear foci. How FANCD2 monoubiquitination is coupled to the DNA-damage response has remained undetermined. This study demonstrates that the ATR checkpoint kinase and RPA1 are required for efficient FANCD2 monoubiquitination. Deficiency of ATR function, either in Seckel syndrome, which clinically resembles Fanconi anemia, or by siRNA silencing, results in the formation of radial chromosomes in response to the DNA cross-linker, mitomycin C (MMC), thus mimicking the chromosome instability of FA cells (Andreassen, 2004).
SN1-type alkylating agents represent an important class of chemotherapeutics, but the molecular mechanisms underlying their cytotoxicity are unknown. Thus, although these substances modify predominantly purine nitrogen atoms, their toxicity appears to result from the processing of O6-methylguanine (6MeG)-containing mispairs by the mismatch repair (MMR) system, because cells with defective MMR are highly resistant to killing by these agents. In an attempt to understand the role of the MMR system in the molecular transactions underlying the toxicity of alkylating agents, the response of human MMR-proficient and MMR-deficient cells to low concentrations of the prototypic methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was studied. MNNG treatment induces a cell cycle arrest that is absolutely dependent on functional MMR. Unusually, the cells arrested only in the second G2 phase after treatment. Downstream targets of both ATM (Ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) kinases were modified, but only the ablation of ATR, or the inhibition of CHK1, attenuated the arrest. The checkpoint activation was accompanied by the formation of nuclear foci containing the signaling and repair proteins ATR, the S*/T*Q substrate, gamma-H2AX, and replication protein A (RPA). The persistence of these foci implies that they may represent sites of irreparable damage (Stojic, 2004).
The Mcm2-7 complex is the catalytic core of the eukaryotic replicative helicase. This study identifies a new role for this complex in maintaining genome integrity. Using both genetic and cytological approaches, it was found that a specific mcm (see Drosophila Mcm2) allele (mcm2DENQ) causes elevated genome instability that correlates with the appearance of numerous DNA-damage associated foci of γH2AX (see Drosophila His2Av) and Rad52 (see Drosophila spn-A). Further, the triggering events for this genome instability were found to be elevated levels of RNA:DNA hybrids and an altered DNA topological state, as over-expression of either RNaseH (an enzyme specific for degradation of RNA in RNA:DNA hybrids) or Topoisomerase 1 (an enzyme that relieves DNA supercoiling) can suppress the mcm2DENQ DNA-damage phenotype. Moreover, the observed DNA damage has several additional unusual properties, in that DNA damage foci appear only after S-phase, in G2/M, and are dependent upon progression into metaphase. In addition, the resultant DNA damage is not due to spontaneous S-phase fork collapse. In total, these unusual mcm2DENQ phenotypes are markedly similar to those of a special previously-studied allele of the checkpoint sensor kinase ATR/MEC1 (see Drosophila mei-41), suggesting a possible regulatory interplay between Mcm2-7 and ATR during unchallenged growth. As RNA:DNA hybrids primarily result from transcription perturbations, the study suggests that surveillance-mediated modulation of the Mcm2-7 activity plays an important role in preventing catastrophic conflicts between replication forks and transcription complexes. Possible relationships among these effects and the recently discovered role of Mcm2-7 in the DNA replication checkpoint induced by HU treatment are discussed (Vijayraghavan, 2016).
ETAA1 (Ewing tumor-associated antigen 1), also known as ETAA16, was identified as a tumor-specific antigen in the Ewing family of tumors. However, the biological function of this protein remains unknown. This study reports the identification of ETAA1 as a DNA replication stress response protein. ETAA1 specifically interacts with RPA (Replication protein A) via two conserved RPA-binding domains and is therefore recruited to stalled replication forks. Interestingly, further analysis of ETAA1 function revealed that ETAA1 participates in the activation of ATR signaling pathway (see Drosophila meiotic 41) via a conserved ATR-activating domain (AAD) located near its N terminus. Importantly, both RPA binding and ATR activation are required for ETAA1 function at stalled replication forks to maintain genome stability. Therefore, these data suggest that ETAA1 is a new ATR activator involved in replication checkpoint control (Lee, 2016).
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