okra

DEVELOPMENTAL BIOLOGY

Embryonic

The RAD54 gene of Saccharomyces cerevisiae plays a crucial role in recombinational repair of double-strand breaks in DNA. DmRAD54 RNA is detected at all stages of fly development, but an increased level was observed in early embryos and ovarian tissue (Kooistra, 1997).

Effects of Mutation or Deletion

To determine the function of DmRAD54, a null mutant was isolated by random mutagenesis. DmRADS4-deficient flies develop normally, but the females are sterile. Early development appears normal, but the eggs do not hatch, indicating an essential role for DmRAD54 in development. The larvae of mutant flies are highly sensitive to X rays and methyl methanesulfonate. Moreover, this mutant is defective in X-ray-induced mitotic recombination as measured by a somatic mutation and recombination test. These phenotypes are consistent with a defect in the repair of double-strand breaks and imply that the RAD54 gene is crucial in repair and recombination in a multicellular organism. The results also indicate that the recombinational repair pathway is functionally conserved in evolution (Kooistra, 1997).

To quantify the ventralization observed in okr, spnB, and spnD mutants, eggshells were assigned to one of four phenotypic classes. Class 1 eggs resemble wild type with two normal dorsal appendages; class v2 eggs have two dorsal appendages that are fused at the base; class v3 eggs have a single dorsal appendage, and class v4 eggs have little or no dorsal appendage material. Females mutant for alleles of okr, spnB, and spnD produce eggs in all four classes, and this variability is not caused by differential expressivity, since a single female produces the full range of phenotypes. Although all three genes give rise to the same range of phenotypes, differences in the distribution of eggs among the various classes reflect the severity of a particular genotype. The spnB and spnD eggshell phenotypes become more severe over time. Newly eclosed spnB and spnD mutant females produce 90%-95% class 1 eggs in the first day after mating, and some of these eggs hatch and give rise to viable progeny. By the fourth day, however, the mutant females produce only 10%-20% class 1 eggs, and there is a corresponding increase in the percentage of class 4 eggs. No change is observed in the severity of the okr eggshell phenotype with time (Ghabrial, 1998).

In addition to the dorsal-ventral patterning defects observed in eggshells, okra mutants share another phenotype with mutants in the grk-Egfr signaling pathway: they produce eggs that often have a second micropyle at the posterior end. This phenotype appears in ~2% of the eggs laid by females homozygous for amorphic okr alleles, and in 42% of the eggs laid by females mutant for the more severe antimorphic alleles. This follicle cell defect can also be visualized with molecular markers: 77% of the egg chambers from strong okr mutations show dpp expression at both the anterior and posterior poles instead of the normal restricted expression in anterior follicle cells. In these mutant ovaries, a defect in Bicoid mRNA localization is observed: 5% of the egg chambers show localization of BCD to both the anterior and posterior poles of the oocyte, indicating that the anterior-posterior polarity of the oocyte is also affected. These data are consistent with the hypothesis that okr affects both the early (anterior-posterior) and late (dorsal-ventral) grk-Egfr signaling processes. However, the appearance of second micropyles on okr mutant eggs does not necessarily reflect the severity of the dorsal-ventral defect: Second micropyles are sometimes observed on eggs with normal dorsal-ventral polarity, and strongly ventralized eggs do not necessarily have a second micropyle. This uncoupling of the two phenotypes implies that okr can affect the early grk signaling process, independent of the later one. In spnB and spnD mutant eggs, significant numbers of second micropyles are not observed, nor are duplications of dpp or mislocalization of bcd seen in the mutant ovaries (Ghabrial, 1998).

A characteristic of mutations in the grk-Egfr signaling pathway is that they affect patterning in both the eggshell and embryo. The embryos that develop within the ventralized eggshells produced by grk and Egfr mutant females are also ventralized, and show an expansion of the mesodermal anlage. To determine if okr, spnB, and spnD affect embryonic patterning as well as eggshell patterning, the expression of the mesodermal marker Twist (Twi) was examined in the mutant embryos. Even though only a small percentage of the mutant embryos develop to the cellular blastoderm stage, those that do develop show a variable expansion of the mesoderm, ranging from cases in which the mesoderm is fairly normal to cases in which it encompasses most of the blastoderm. Notably, this expansion is always more severe at the posterior than the anterior. In addition to these phenotypes, one also sees cases in which the mesoderm is normal at the anterior end of the embryo, but which at the posterior splits into two independent domains that run up the lateral sides of the embryo and meet at the dorsal midline. Apart from the difference in ventralization along the anterior-posterior axis, these ventralized phenotypes are similar to those that have been observed in grk and Egfr mutant embryos, and suggest that okr, spnB, and spnD affect dorsal-ventral patterning via an effect on grk-Egfr signaling (Ghabrial, 1998).

In yeast, components of the RAD52 epistasis group are required for the recombinational repair of double stranded breaks (DSBs) in both mitotic and meiotic cells. In mitotic cells, mutations in these genes interfere with the cell's ability to repair DNA damage, whereas in meiotic cells, they block genetic recombination resulting from the failure to repair DSBs associated with crossing over. In light of the homology of okr and spnB to genes in this epistasis group, a determination was made of whether mutations in okr, spnB, and spnD affect mitotic and meiotic DSB repair. To look for a requirement in mitotic DSB repair, various mutant genotypes were tested for sensitivity to DNA damage. To look for a requirement in meiotic DSB repair, mutant genotypes were tested for a reduction in meiotic exchange. To test for sensitivity to DNA damage, crosses producing okr, spnB, and spnD mutant larvae were fed a solution of 0.08% methylmethanesulfonate (MMS), a chemical mutagen that induces DSBs. The survival of MMS-treated larvae was compared with that of mutant larvae from an untreated control cross. okr mutants are found to be sensitive to MMS, showing a significant reduction in survival in MMS-treated crosses relative to control crosses. In contrast, spnB and spnD mutants are not sensitive, showing equal percentages of expected progeny in both crosses. The fact that spnB and spnD mutants do not show MMS sensitivity suggests that they may not be required for mitotic DSB repair (Ghabrial, 1998).

To test the effect of okr, spnB, and spnD mutations on meiotic DSB repair, the effects of these mutants was measured on meiotic exchange as reflected in the frequency of recombination and X-chromosome nondisjunction. For these experiments, advantage was taken of the fact that females mutant for even the strongest spnB and spnD alleles produce escaper progeny (progeny that survive to adulthood) in the first days after mating. In the case of okr, it was not possible to use the strongest alleles because they are almost completely sterile. Instead, a weak allele, okrAO, was used, which is fertile as a homozygote and hemizygote (25% and 50% hatching, respectively). In spnB or spnD mutant females heterozygous for X chromosomal markers, the frequency of recombination is 10%-25% of normal levels, whereas for the weak okr allele the frequency of recombination is at 50% of normal levels. In crosses that allowed the scoring of the exceptional progeny classes produced by X chromosome nondisjunction, an ~100-fold increase in X chromosome nondisjunction was observed in both spnB and spnD mutant females, as well as a 17- to 20-fold increase in the crosses involving okr. Although the results for okr are not as dramatic as those for spnB and spnD, it is likely that stronger okr alleles would show a more severe effect. In summary, the data are consistent with a requirement for spnB, spnD, and okr in meiotic DSB repair (Ghabrial, 1998).

A hallmark of germline cells across the animal kingdom is the presence of perinuclear, electron-dense granules called nuage. In many species examined, Vasa, a DEAD-box RNA helicase, is found in these morphologically distinct particles. Despite its evolutionary conservation, the function of nuage remains obscure. A null allele of maelstrom (mael) has been characterized. Maelstrom protein is localized to nuage in a Vasa-dependent manner. By phenotypic characterization, maelstrom has been defined as a spindle-class gene that affects Vasa modification. In a nuclear transport assay, it has been determined that Maelstrom shuttles between the nucleus and cytoplasm, which may indicate a nuclear origin for nuage components. Interestingly, Maelstrom, but not Vasa, depends on two genes involved in RNAi phenomena for its nuage localization: aubergine and spindle-E (spn-E). Furthermore, maelstrom mutant ovaries show mislocalization of two proteins involved in the microRNA and/or RNAi pathways, Dicer and Argonaute2, suggesting a potential connection between nuage and the microRNA-pathway (Findley, 2003).

How germline status is established and maintained in sexually reproducing organisms is a fundamental question in developmental biology. A conserved feature of germ cells in species across the animal kingdom is the presence of a distinct morphological element called nuage. Ultrastructurally, nuage appears as electron-dense granules that are localized to the cytoplasmic face of the nuclear envelope. Despite the breadth of nuage in the animal kingdom, there is currently a lack of depth in understanding its function. In animals ranging from the nematode to vertebrates, the Vasa protein has been detected in these granules. Both nuage and Vasa thus offer potential clues as to what makes a germ cell unique (Findley, 2003).

One system with high potential for understanding the role of nuage is Drosophila. In females, Vasa-positive germline granules are continuously present throughout the life cycle, taking one of two forms, nuage or pole plasm. Pole plasm, which contains polar granules, is a determinant that is both necessary and sufficient to induce formation of the germ lineage in early embryogenesis. In Drosophila, nuage is first detectable when primordial germ cells are formed; it persists through adulthood, where it is present in all germ cell types of the ovary (Findley, 2003).

A null allele of the maelstrom gene, which encodes a novel protein with a human homolog, has been identified and characterized. The mutant displays each of the defects in oocyte development common to the spindle-class. Maelstrom localizes to nuage in a Vasa-dependent manner and maelstrom is required for proper modification of Vasa. Through mutant analysis, this study begins to unravel genetic dependencies of nuage particle assembly (Findley, 2003).

Spn-E encodes a putative Dex/hD-box RNA helicase, required for proper localization of several oocyte-destined RNAs and proteins over the course of oogenesis. While the localization of Spindle-E in the ovary has not been determined, its involvement in both RNAi and oogenesis, like Aubergine, prompted its inclusion in this analysis. As with aubergine mutants, the concentration of Maelstrom in perinuclear particles is lost in strong spn-E allelic combinations, spn-E^{616/hlsDelta125} and spn-E^{hls3987/hlsDelta125}. Vasa retains a perinuclear concentration in spn-E ovaries, but as in aubergine, the normal particulate appearance of nuage is less pronounced. Localization analysis has been extended to include the remaining members of the better characterized spn-class mutants, spn-A, spn-B, spn-C, spn-D and okr. Of particular interest was spn-B, which has been shown to modify Vasa as a consequence of meiotic checkpoint activation. The dependency of Maelstrom on Vasa for its localization could, in principle, be affected if Vasa is aberrant. However, in multiple allelic combinations of well-characterized spn genes (spn-B, spn-D and okr) and uncloned spn genes (spn-A and spn-C), colocalization of Vasa and Maelstrom in nuage particles was unperturbed at all stages of oogenesis (Findley, 2003).

The dissociation of Maelstrom from nuage particles in aubergine and spn-E backgrounds was intriguing in light of their requirement in RNAi in Drosophila spermatogenesis and late oogenesis. Importantly, proteins (or homologs) of RNAi pathway components also act in micro RNA (miRNA) processing. Since miRNAs have been shown to regulate RNA translation, it is conceivable that miRNAs are assembled in RNP particles formed in nuage. In this setting, nuage could represent a step in the generation of specificity in translational control in the germline. To explore this potential relationship between nuage and RNAi/miRNA processing pathways, the localization of additional RNAi components was examined in wild-type and maelstrom ovaries. Argonaute1 and Argonaute2 are RDE1/AGO1 homologs required for RNAi in Drosophila. Dicer is the core RNase of RNAi in Drosophila; it is also required for production of the small RNA effectors of the RNAi and miRNA pathways in C. elegans. In vertebrate cell lines, Dicer is primarily cytoplasmic. In wild-type Drosophila ovarioles, Dicer and AGO1 appear uniform and cytoplasmic in nurse cell cytoplasm; AGO2 appears cytoplasmic but relatively more granular. In maelstrom ovaries, AGO1 distribution is relatively unperturbed. However, AGO2 and Dicer are both dramatically mislocalized in maelstrom ovarioles. Beginning around stage 3, Dicer aggregates in discrete, often perinuclear foci in nurse cells. AGO2 is observed in perinuclear regions of nurse cells, which, by contrast, can colocalize with Vasa in nuage (Findley, 2003).

The failure of maelstrom oocytes to proceed to the karyosome stage, to establish cytoplasmic polarity and to accumulate Gurken qualifies the inclusion of maelstrom in the spindle class. Maelstrom is a component of Drosophila nuage and is required for proper modification (or processing) of a key nuage component, Vasa. Maelstrom is also present within the nucleus and cytoplasm of all germline cells, and can shuttle between these compartments in a CRM1-dependent manner. Of the known nuage-localizing proteins, Vasa appears to be a pivotal organizer or nucleator of nuage, whereas Maelstrom can be dissociated from nuage particles in aubergine and spn-E mutants. Furthermore, Dicer and AGO2 are mislocalized in the maelstrom background (Findley, 2003).

The characterized spn genes currently fall into two general classes: those that encode proteins that are likely to be directly involved in meiotic recombinational repair, such as okr, spn-B and spn-C; and those, such as maelstrom and vasa, whose mutant meiotic phenotype, protein sequence and/or localization suggest indirect roles. Work presented in this study suggests that the spn mutants can be sorted by an additional criterion: those that are also required for nuage assembly (vasa, aubergine, maelstrom and spn-E) and those that are not (spn-A, spn-B, spn-C, spn-D and okra). Taken together, these data suggest that the Vasa-like group of spn genes are essential in general 'nuage activities' in all cells of the germline. The activity of the spn-B-class genes, which are involved in recombination or meiotic checkpoint, could represent one avenue through which to use or modulate existing nuage functions that are operative within the germline cyst as a whole. Such nuage-related processes, if inactivated or defective, might culminate in polarity and translational defects within the oocyte (Findley, 2003).

During Drosophila oogenesis, unrepaired double-strand DNA breaks activate a mei-41-dependent meiotic checkpoint, which couples the progression through meiosis to specific developmental processes. This checkpoint affects the accumulation of Gurken protein, a transforming growth factor alpha-like signaling molecule, as well as the morphology of the oocyte nucleus. However, the components of this checkpoint in flies have not been completely elucidated. A mutation in the Drosophila Chk2 homolog (DmChk2/Mnk or loki) has been shown to suppress the defects in the translation of gurken mRNA and also the defects in oocyte nuclear morphology. Drosophila Chk2 is phosphorylated in a mei-41-dependent pathway. Analysis of the meiotic cell cycle progression shows that the Drosophila Chk2 homolog is not required during early meiotic prophase, as has been observed for Chk2 in C. elegans. The activation of the meiotic checkpoint affects Wee localization and is associated with Chk2-dependent posttranslational modification of Wee. It is suggested that Wee has a role in the meiotic checkpoint that regulates the meiotic cell cycle, but not the translation of gurken mRNA. In addition, p53 and mus304, the Drosophila ATR-IP homolog, are not required for the patterning defects caused by the meiotic DNA repair mutations. It is concluded that Chk2 is a transducer of the meiotic checkpoint in flies that is activated by unrepaired double-strand DNA breaks. Activation of Chk2 in this specific checkpoint affects a cell cycle regulator as well as mRNA translation (Abdu, 2002).

In the budding yeast, checkpoint-dependent cell cycle arrest at pachytene is achieved by the accumulation of hyperphosphorylated Swe1p, a Wee1-like protein, and subsequent inactivation of Cdc28p. Like other metazoans, Drosophila has two Wee1-like kinases, Wee and Dmyt1. To study the role of Wee in the meiotic checkpoint, the Wee expression in ovaries from spindle-class mutants was compared to expression in wild-type by using an anti-Wee antibody. Western blot analysis shows that the mobility of Wee1 protein is retarded in spn-B, okr, and spn-D mutant ovaries. Wee1 protein also migrates slowly in ovarian extracts prepared from flies mutant for spn-B and grp. In contrast, the mobility of Wee in flies mutant for spn-B and Chk2 is restored to wild-type. Immunohistochemical assays also show an abnormal Wee subcellular localization in spindle-class genes. In wild-type ovaries, Wee protein accumulates inside the oocyte nucleus but is excluded from the DNA, whereas, in about 37% of mutant egg chambers from spn-B, okra, and spn-D, Wee protein accumulates throughout the oocyte nucleus. Interestingly, it was found that mutations in Wee are not able to suppress the dorsal-ventral patterning or the oocyte nuclear morphology defects caused by mutations in the spindle-class genes. Expression of an active form of Cdc2 alone or together with Cyclin A in spn-B mutant flies does not suppress these defects (Abdu, 2002).

The changes in the Wee expression in spindle-class mutants suggest that the initiation of the meiotic checkpoint affects the meiotic cell cycle progression in a Wee-dependent manner, as it does in yeast. However, mutations in Wee1 do not suppress the patterning defects in spindle-class mutants. It is possible that two different pathways are activated by the persistence of unrepaired double-strand DNA breaks, one affecting Wee and the cell cycle, and a second pathway leading to Vasa modification and patterning defects. Alternatively, it is possible that other cell cycle regulators act in parallel to Wee and that the primary effect of the checkpoint is cell cycle arrest, which in turn affects Vasa modification and patterning. However, several studies suggest that, in spindle mutants, there is only a transient cell cycle arrest during early oogenesis, whereas the major effect on translation of grk mRNA occurs during mid-oogenesis. Thus, it is proposed that the patterning defects in spindle mutants are not the result of checkpoint-induced cell cycle arrest (Abdu, 2002).

In summary, the results demonstrate that the Drosophila Chk2 homolog is a transducer of the meiotic checkpoint that is activated by unrepaired double-strand DNA breaks. Activation of Chk2 results in modification of two proteins, Vasa and Wee, which then affect progression of the meiotic cell cycle and translation of gurken mRNA. Wee is, however, not required for the patterning defects seen in the spindle mutations. Activation of the Chk2-dependent meiotic checkpoint may therefore control several cell cycle regulators which in turn may affect both meiosis and translation of gurken mRNA. In particular, it is likely that Wee1 activation regulates cell cycle progression, whereas Chk2 may utilize an independent target to regulate Vasa, which subsequently affects dorsal-ventral patterning as well as nuclear morphology of the oocyte. While dorsal-ventral signaling by Gurken is not a conserved feature of oogenesis found in other organisms, the fact that homologs of Drosophila Chk2 act during meiosis in other organisms raises the possibility that meiotic checkpoints in other species might also act through Chk2 to regulate translation during oogenesis and thus directly link the meiotic cell cycle to the development of the oocyte (Abdu, 2002).

The relationship between synaptonemal complex formation (synapsis) and double-strand break formation (recombination initiation) differs between organisms. Although double-strand break creation is required for normal synapsis in Saccharomyces cerevisiae and the mouse, it is not necessary for synapsis in Drosophila and Caenorhabditis elegans. To investigate the timing of and requirements for double-strand break formation during Drosophila meiosis, an antibody was used that recognizes a histone modification at double-strand break sites: phosphorylation of HIS2AV (gamma-HIS2AV). The results support the hypothesis that double-strand break formation occurs after synapsis. Interestingly, a low (10%-25% of wildtype) number of gamma-HIS2AV foci has been detected in c(3)G mutants, which fail to assemble synaptonemal complex, suggesting that there may be both synaptonemal complex-dependent and synaptonemal complex-independent mechanisms for generating double-strand breaks. Furthermore, mutations in Drosophila Rad54 (okr) and Rad51 (spnB) homologs cause delayed and prolonged gamma-HIS2AV staining, suggesting that double-strand break repair is delayed but not eliminated in these mutants. There may also be an interaction between the recruitment of repair proteins and phosphorylation (Jang, 2003).

Meiotic recombination in Drosophila melanogaster requires the tropoisomerase-like mei-W68, a Spo11 homolog. On the basis of physical studies and homology to the archaea topoisomerase II subunit Top6A, Spo11 is presumed to be the protein responsible for double-strand break (DSB) formation (and therefore meiotic recombination initiation) in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Furthermore, Spo11 homologs have been identified in a wide variety of organisms, leading to the hypothesis that the creation of DSBs is a conserved mechanism for initiating meiotic recombination. Similarly, repair of meiotic DSBs requires members of a conserved group of genes such as homologs of Rad51 and Rad54 (Jang, 2003).

Another hallmark of meiotic prophase is the synaptonemal complex (SC), a specialized protein-chromosome structure that physically connects aligned homologous chromosomes. Completion of synapsis between homologous chromosomes is marked by the presence of SC along their entire lengths. SC can form in the absence of DSBs in Drosophila and C. elegans, but not in budding yeast. The relative timing of DSBs and SC formation in the mouse was determined using an antibody that recognizes the phosphorylated form of a histone H2A variant, H2AX. On induction of DSBs in mammalian mitotic and meiotic cells, H2AX is rapidly phosphorylated. gamma-H2AX staining is detected before the appearance of SC proteins, suggesting that DSBs appear before SC formation during meiotic prophase in the mouse, consistent with time-course studies of DSB formation in S. cerevisiae (Jang, 2003).

Drosophila has a single H2A variant (HIS2AV) that, like H2AX, is phosphorylated at a conserved SQ motif within an extended C-terminal tail following chromosome breakage in mitotic cells. Phosphorylation of Drosophila HIS2AV (gamma-HIS2AV) in mitotic cells is rapid, reaching its maximum within five minutes of exposure to agents that induce DSBs but not single-strand nicks. Evidence is provided that HIS2AV is phosphorylated in response to meiotic DSB formation. Using gamma-HIS2AV staining as a marker for DSB formation, evidence was found that DSB formation occurs after synapsis and is partially dependent on the SC protein C(3)G. In addition, gamma-HIS2AV staining suggests that DSB repair is delayed in okr (Rad54 homolog) and spnB (Rad51/Dmc1 homolog) mutants, but it does occur eventually (Jang, 2003).

In wild-type female meiosis, the gamma-H2AX antibody detected nuclear foci in early pachytene cells (region 2a and occasionally region 2b of the germarium). The rate of HIS2AV phosphorylation cannot be directly measured in Drosophila meiotic cells, but in somatic cells, phosphorylation reaches a maximum within 5 minutes of exposure to agents that induce DSBs and is almost completely removed within 3 hours. Given that the life-span of gamma-HIS2AV foci is likely to be short relative to the developmental age difference between each cyst in the germarium (~12 hours), the foci could appear at DSBs that are induced early in pachytene (region 2a) and disappear later in pachytene (regions 2b and 3) because of repair (Jang, 2003).

Consistent with the hypothesis that the foci disappear because of DSB repair, a dramatic change in the gamma-HIS2AV staining pattern was observed in spnB (a meiosis-specific Rad51 homolog) and okr (a Rad54 homolog) mutants, which are proposed to be defective in DSB repair. Although the wild-type gamma-HIS2AV foci are limited to early pachytene (region 2a and occasionally 2b) and are always absent from late pachytene oocytes (region 3), the okr^WS and spnB^BU mutant germaria always exhibit foci in late pachytene (region 3 and early vitellarium). In addition, the number of gamma-HIS2AV foci in region 3 oocytes of the DSB repair-defective mutants was consistent and usually higher than in wildtype, often in excess of 20 gamma-HIS2AV foci. The foci persisted in the okr and spnB mutants until stage 4 of the vitellarium, at which point they disappeared, just before the dissolution of the SC. The persistent and numerous foci in these mutants may result from the failure to efficiently repair DSBs. Indeed, the gamma-HIS2AV staining in okr^WS and spnB^BU mutants gradually becomes brighter until region 3, suggesting that the gamma-HIS2AV is accumulating (Jang, 2003).

The Ligase IV gene plays a crucial role in the repair of radiation-induced DNA double-strand breaks and acts synergistically with Rad54

DNA Ligase IV has a crucial role in double-strand break (DSB) repair through nonhomologous end joining (NHEJ). Most notably, its inactivation leads to embryonic lethality in mammals. To elucidate the role of DNA Ligase IV (Lig4) in DSB repair in a multicellular lower eukaryote, viable Lig4-deficient Drosophila strains were generated by P-element-mediated mutagenesis. Embryos and larvae of mutant lines are hypersensitive to ionizing radiation but hardly so to methyl methanesulfonate (MMS) or the crosslinking agent cis-diamminedichloroplatinum (cisDDP). To determine the relative contribution of NHEJ and homologous recombination (HR) in Drosophila, Lig4; Rad54 double-mutant flies were generated. Survival studies have demonstrated that both HR and NHEJ have a major role in DSB repair. The synergistic increase in sensitivity seen in the double mutant, in comparison with both single mutants, indicates that both pathways partially overlap. However, during the very first hours after fertilization NHEJ has a minor role in DSB repair after exposure to ionizing radiation. Throughout the first stages of embryogenesis of the fly, HR is the predominant pathway in DSB repair. At late stages of development NHEJ also becomes less important. The residual survival of double mutants after irradiation strongly suggests the existence of a third pathway for the repair of DSBs in Drosophila (Gorsky, 2003).

To counteract the deleterious effects of DSBs, two main repair pathways exist in eukaryotes: homologous recombination (HR) and nonhomologous end joining (NHEJ). HR requires the presence of an undamaged homologous DNA that can be used as a template. In this way, HR ensures accurate DSB repair. NHEJ is based on ligation of the two ends and does not require extensive sequence homology. Frequently, NHEJ is associated with insertion or deletion of a few nucleotides at the site of the break. The relative contribution of both repair pathways depends on the organism, the phase of the cell cycle, the developmental stage, and, presumably, the structure of the break that forms. In lower eukaryotes, HR is the primary repair mechanism. In the yeast Saccharomyces cerevisiae, the contribution of the NHEJ to the repair of DSBs can be detected only when HR is impaired. In higher eukaryotes, both HR and NHEJ contribute to the repair of DSBs. HR is especially important during the S and G2 phases of the cell cycle when the sister chromatid can be used as a template and during early development. NHEJ predominates in adult organisms and during the G1 and early S phases of the cell cycle. The NHEJ pathway was first studied in mammals using rodent cell mutants and involves a number of proteins including Ku70, Ku80, DNA-PKcs, Ligase IV and its associated protein XRCC4, and the Artemis protein. The current model of DSB repair by NHEJ assumes that a heterodimer of Ku70 and Ku80 binds to DNA ends and recruits DNA-PKcs to the site of the damage to form an active DNA-PK complex. Binding of Ku to the DNA ends is also required for recruitment of Ligase IV and XRCC4 to the site of the break. This recruitment results in stimulation of DNA end ligation. The newly identified Artemis protein binds to DNA-PKcs and has endo- and exonucleolitic activities required for processing of DNA ends (Gorsky, 2003 and references therein).

Mice deficient in one of the components of the DNA-PK complex display an increased sensitivity to ionizing radiation and a severe combined immunodeficiency phenotype due to defects in V(D)J recombination. Mouse embryonic stem cells deficient in DNA-PKcs, however, are not hypersensitive to ionizing radiation. Evidently, the repair of DSBs by NHEJ is independent of DNA-PKcs in these cells. In yeast and also in Drosophila and Caenorhabditis elegans, no obvious homolog of DNA-PKcs has been identified. The role of DNA-PKcs possibly could be restricted to cells that strongly rely on NHEJ for the repair of X-ray-induced DSBs. In contrast to the loss of DNA-PKcs or the Ku function, inactivation of Ligase IV or XRCC4 in mice results in embryonic lethality as a consequence of massive apoptosis in the central nervous system. Rescue of embryonic lethality is possible in a p53-, ATM-, or Ku-deficient background. In addition to the repair of DSBs, studies in yeast and mammalian cells indicate that the Ku proteins and DNA-PKcs are also involved in maintenance of telomere length and normal chromosomal DNA end structure (Gorsky, 2003 and references therein).

Drosophila has been used extensively to study the mutagenic effects of ionizing radiation (IR) and it represents an attractive system to study DSB repair in a multicellular organism. Flies deficient in Rad54 are highly sensitive to X rays and methyl methanesulfonate (MMS), implying that HR contributes significantly to the repair of DSBs in somatic cells. Inactivation of Rad54 or spindle-B, one of the Rad51 paralogs in Drosophila, leads to defects in meiosis. Increased MMS sensitivity, as compared with wild-type strains, has not been observed for the spindle-B mutant (Gorsky, 2003).

To study the contribution of NHEJ to the repair of DSBs in flies and to investigate the role of DNA Ligase IV in a multicellular organism, the Drosophila DNA Ligase IV gene, Lig4 was isolated, and its function was examined by generating mutant strains. In contrast to mice, homozygous null flies are viable and show increased sensitivity to ionizing radiation. A strong synergistic effect for radiosensitivity was detected in Lig4; Rad54 double-mutant flies (Gorsky, 2003).

The survival data of double-mutant flies demonstrate that in Drosophila both NHEJ and HR contribute significantly to the repair of DSBs induced by ionizing radiation. The data also indicate that with the exception of 0- to 4-hr embryos, both mechanisms can partially compensate for each other. At later stages of development (48-96 hr) the analysis of the double mutant suggests a less important role for NHEJ. NHEJ and HR have been presented as competing pathways. Binding of Ku or Rad52 proteins to DNA ends at the site of the break would initiate DSB repair through NHEJ or HR, respectively. The result of such a competition is influenced by the relative amount of Ku70 and Rad52 (or by a functionally related protein in Drosophila, since a structural Rad52 homolog has not been identified), structure of the DSB, cell cycle phase, and stage of development. The pathway that is used has important consequences for the integrity of the genetic information of an organism. NHEJ is frequently associated with loss or gain of a few nucleotides. Correct restoration of the original sequence can occur via HR if the sister chromatid is used as a template. Using the homologous chromosome as a template could lead to loss of heterozygosity. Early embryonic development in Drosophila is a very rapid process. After fertilization the zygote nucleus undergoes nine divisions in a common cytoplasm to produce a multinucleate syncytium. After migration to the periphery of the egg, the nuclei undergo four more divisions before a cellular membrane is formed and somatic cells are produced. This process takes only 2.5 hr. To avoid accumulation of mutations during the rapid early divisions, which may have deleterious consequences at adult stages, it is beneficial to use HR as the principal mechanism in early development. Studies in mice also indicate that HR is especially important in early development. In contrast to mouse embryonic stem cells, a contribution of NHEJ cannot be detected in 0- to 4-hr-old Drosophila embryos, although the possibility that defects in NHEJ can be fully compensated by HR in contrast to later stages cannot be excluded. After the first 4 hr of embryonic development NHEJ does play an important role in the repair of DSBs. Between 4 and 20 hr of development Lig4-deficient flies are most sensitive to increasing doses of ionizing radiation. The hypersensitivity of Lig4-deficient larvae to IR is gradually reduced at later stages of development, indicating that the majority of the radiation-induced DSBs are repaired by HR and only a small fraction by NHEJ. It is difficult to speculate whether it is a competition between the repair pathways that causes those shifts or whether yet another repair system is active at later stages. Later in development the cell divisions definitely become much slower so it is not a matter of cell cycle stage and/or template availability, which would preferentially shift the repair toward HR. These observations differ from the data obtained from mouse studies. Mice deficient for RAD54 are hypersensitive only at very early embryonic stages. In adult mice no hypersensitivity to ionizing radiation was seen in contrast to mice deficient in NHEJ (Gorsky, 2003).

The viability of the Lig4; Rad54 mutant flies, as well as survival after low levels of X-ray irradiation, could be explained by evasion of checkpoint control and/or escape from checkpoint-triggered apoptosis at certain stages of the cell cycle or of development. Another possibility is that undamaged dividing cells in the imaginal discs can compensate for the loss of damaged and/or apoptotic cells. The viability of the double mutant after irradiation could also suggest the presence of another repair pathway that partially compensates for the impaired HR and NHEJ mechanisms. One possibility is single-strand annealing (SSA). This mechanism relies on the annealing of repeated sequences on both sides of the DNA break after the formation of 3'-single-strand tails. Evidence exists for the existence of SSA in Drosophila. Another mechanism that possibly can overcome IR-induced DSBs in double-mutant flies is microhomology-dependent end joining (µEJ). Evidence for the existence of this pathway has been obtained from studies of mammalian cells mutated in one of the components required for NHEJ. Studies in yeast using engineered substrates transfected into ku70/rad52 or ku80/rad52 double mutants indicate the existence of a repair mechanism, which repairs DSBs on the basis of microhomology present on both sides of the break. Since small repeated sequences are frequently associated with the formation of chromosomal aberrations in mammalian cells, it will be of great interest to investigate the µEJ pathway in more detail in eukaryotic organisms, including Drosophila. The availability of Lig4 and Rad54 single and double mutants allows further studies into the mechanisms of HR and NHEJ to be pursued (Gorsky, 2003).

Relative contribution of DNA repair, cell cycle checkpoints, and cell death to survival after DNA damage in Drosophila larvae

Components of the DNA damage checkpoint are essential for surviving exposure to DNA damaging agents. Checkpoint activation leads to cell cycle arrest, DNA repair, and apoptosis in eukaryotes. Cell cycle regulation and DNA repair appear essential for unicellular systems to survive DNA damage. The relative importance of these responses and apoptosis for surviving DNA damage in multicellular organisms remains unclear. After exposure to ionizing radiation, wild-type Drosophila larvae regulate the cell cycle and repair DNA; grp (DmChk1) mutants cannot regulate the cell cycle but repair DNA; okra (DmRAD54) mutants regulate the cell cycle but are deficient in repair of double strand breaks (DSB); mei-41 (DmATR) mutants cannot regulate the cell cycle and are deficient in DSB repair. All undergo radiation-induced apoptosis. p53 mutants regulate the cell cycle but fail to undergo apoptosis. Of these, mutants deficient in DNA repair, mei-41 and okra, show progressive degeneration of imaginal discs and die as pupae, while other genotypes survive to adulthood after irradiation. Survival is accompanied by compensatory growth of imaginal discs via increased nutritional uptake and cell proliferation, presumably to replace dead cells. It is concluded that DNA repair is essential for surviving radiation as expected; surprisingly, cell cycle regulation and p53-dependent cell death are not. It is proposed that processes resembling regeneration of discs act to maintain tissues and ultimately determine survival after irradiation, thus distinguishing requirements between muticellular and unicellular eukaryotes (Jaklevic, 2004).

In eukaryotes, DNA damage checkpoints monitor the state of genomic DNA and delay the progress through the cell cycle as needed. Central components of this checkpoint in mammals include four kinases: ATM, ATR, Chk1, and Chk2. Homologs of these exist in other eukaryotes and assume similar roles where examined. Human patients with ATM mutations, as well as their cells, show a dramatic sensitivity to killing by ionizing radiation. The importance of checkpoints in cellular survival to DNA damaging agents is presumed to be due to the role of checkpoints in cell cycle regulation. This is because mutants in the budding yeast gene rad9, the first checkpoint gene to be characterized, fail to arrest the cell cycle following damage and show increased radiation sensitivity; the latter phenotype is rescued by experimental induction of cell cycle delay. Consequently, cell cycle delay is thought to allow time for DNA repair and thereby ensure survival (Jaklevic, 2004 and references therein).

Components of the DNA damage checkpoint are found to activate DNA repair and to promote programmed cell death, which would cull cells with damaged DNA. For example, phosphorylation of NBS (a component of the Mre11 repair complex) by human ATM is of functional importance, while ATM knockout mice show a reduction in radiation-induced cell death in the CNS. Therefore, the essential role of checkpoints in conferring survival to genotoxins may be due to DNA repair and cell death responses in addition to or instead of cell cycle regulation. Furthermore, what is important for survival at the cellular level may not be so in a multicellular context. For instance, the failure to arrest the cell cycle by checkpoints may be detrimental to individual cells, but removal of these by cell death and replacement via organ homeostasis may make cell cycle regulation inconsequential for survival of multicellular organs (Jaklevic, 2004).

To address how DNA damage checkpoints operate in the context of multicellular organisms in vivo, the effect of ionizing radiation on Drosophila melanogaster is being studied. In Drosophila, mei-41 (ATR homolog) and grp (Chk1 homolog) are required to delay the entry into mitosis in larval imaginal discs after irradiation and to delay the entry into mitosis after incomplete DNA replication in the embryo. Thus, mei-41 and grp play similar roles to their homologs in other systems. Moreover, mei-41 mutants are deficient in DNA repair. The role of mei-41 and grp in radiation-induced cell death has not been tested, but mei-41 is dispensable for cell death after enzymatic induction of DNA double-strand breaks (Jaklevic, 2004 and references therein).

Mutants in mei-41, grp, p53, and okra, a homolog of budding yeast RAD54 that functions in repair of DNA double-strand breaks (DSB) have been used to address the relative importance of cell cycle regulation, cell death, and DNA repair to the ability of a multicellular organism to survive ionizing radiation. The three responses are affected to different degrees in these mutants: wild-type larvae regulate S and M phases and repair DNA; grp mutants are unable to regulate the cell cycle but are able to repair DNA; okra mutants are able to regulate the cell cycle but are deficient in DNA repair; and mei-41 mutants are unable to regulate the cell cycle and are also deficient in DNA repair. All genotypes with the exception of p53 mutants are proficient in radiation-induced cell death, suggesting that mei-41 and grp do not contribute to this response. Under these conditions, it is found that while mei-41 and okra mutants are highly sensitive to killing by ionizing radiation, p53 mutants show reduced but significant survival and grp mutants resemble wild-type. These results suggest that cell death is neither sufficient nor absolutely necessary, DNA repair is essential, and optimal cell cycle regulation is dispensable for surviving ionizing radiation in Drosophila larvae (Jaklevic, 2004).

The effects of DNA damage by ionizing radiation on the maintenance and survival of Drosophila larvae was studied. Despite an extensive loss of cells to radiation-induced cell death, organ size and morphology are maintained remarkably well, and larvae survive to produce viable adults. Surprisingly, optimal cell cycle regulation by checkpoints is neither necessary (as in grp mutants) nor sufficient (as in okra mutants) to ensure organ homeostasis and organismal survival. p53-dependent cell death is also largely dispensable in this regard. Instead, DNA repair appears to be of paramount importance as might be expected (Jaklevic, 2004).

In mitotically proliferating cells of Drosophila larval imaginal discs and brains, the first responses to sublethal doses of irradiation (1000R-4000R) are delays in cell cycle progression at 1-2 hr after irradiation, followed by the induction of cell death at 4 hr after irradiation. DNA synthesis resumes at 5 hr after irradiation, while mitotic index resumes at 6 hr after. These relatively early responses are followed by an increase in proliferation that is detectable about a day after irradiation. Presumably, abundant cell death removes damaged cells, but sustained proliferation compensates to maintain proper organ size and morphology. Continued cell proliferation, it is proposed, delays the onset of the next major developmental transition, pupariation. The extent of delay correlates with radiation dose, presumably because more cells are lost at higher doses, requiring more compensatory proliferation (Jaklevic, 2004).

Another response monitored was DNA repair, a substantial portion of which must occur within 3 hr after 220R of irradiation because a significant difference is seen in the incidence of chromosome breakage between wild-type and repair-deficient mutants by this time. However, cytologically visible chromosome breaks likely represent only a fraction of total DNA damage; for this reason, it is not certain if DNA repair is complete within this time frame (Jaklevic, 2004).

Having determined the sequence of responses to irradiation in wild-type larvae, deviations from it in various mutants was documented. mei-41 and grp mutants are unable to dampen DNA synthesis after irradiation. Previous work has shown that both mutants are unable to inhibit mitosis after irradiation, although grp mutants appear to retain a partial activity in this regard. Thus, Drosophila ATR and Chk1 are needed for optimal regulation of both S and M phases after exposure to ionizing radiation. However, induction of cell death does not require mei-41 or grp. The most striking result report in this study is that grp mutants that are defective in regulation of both S and M phases are not sensitive to killing by 2000R of X-rays, doses that readily killed mei-41 and okra mutants. This finding strongly suggests that cell cycle regulation by checkpoints is not absolutely necessary for surviving irradiation under these conditions (Jaklevic, 2004).

In determining what is necessary, the phenotype of okra mutants that can regulate both S and M phases and promote cell death is particularly informative because they are radiation sensitive. Thus, DNA repair is essential, suggesting that it is this defect in mei-41 mutants that renders them radiation sensitive. It is speculated that irradiated mei-41 and okra larvae may attempt to increase proliferation, but the continual presence of unrepaired DNA likely channels these cells to death. This would lead to an eventual decline in cell number, which would undermine maintenance of cellular differentiation that is the basis of the eye disc's morphogenetic furrow (MF). Signals from cells in the MF are thought to be important for the generation of the second mitotic wave. Loss of the MF could then explain the absence of the expected pattern of mitoses in mei-41 and okra discs (Jaklevic, 2004).

Traditionally, checkpoints refer to the regulation of the cell cycle. Recent views propose the inclusion of the other responses among checkpoint responses, such as the preservation of DNA replication intermediates, transcriptional activation, and DNA repair. The data suggest that other responses may be more important in ensuring survival of multicellular organs and organisms. Interestingly, results from budding yeast also question the idea that cell cycle regulation by checkpoints is essential for surviving genotoxins even at the cellular level. For example, yeast Chk1 mutants show profoundly defective regulation of mitosis after irradiation and yet are only mildly radiation sensitive. Another recent study indicates that stabilization of replication forks is crucial for surviving the alkylating agent MMS whereas the ability to inhibit mitosis is less important (Jaklevic, 2004).

It is emphasized that survival in this study refers to that of organs and organisms. At the cellular level, cell cycle regulation by checkpoints may well be crucial to allow time for DNA repair and for survival. In grp mutants that are defective for cell cycle checkpoints but are proficient for DNA repair, cells that progressed through S and M phases with damaged DNA may have been subject to cell death. Indeed, incidence of cell death appears higher in grp (and mei-41) mutants than in wild-type. Loss of these cells, however, is clearly of little consequence to survival of imaginal discs and larvae. This could be because grp mutants are able to repair DNA in cells that are not in S and M phases, i.e., those in G1 or G2. These cells may then proliferate to compensate for lost cells. Numerous studies on tissue regeneration demonstrate the power of Drosophila larvae to restore not only cell number but also proper differentiation. In such a system, the failure of cell cycle checkpoints after irradiation may be of little consequence as long as damaged cells are replaced. It is speculated that these findings may be particularly applicable to multicellular systems with similar regenerative powers such as the human liver (Jaklevic, 2004).

Lig4 and Rad54 are required for repair of DNA double-strand breaks induced by P-element excision in Drosophila

Site-specific double-strand breaks (DSBs) were generated in the white gene located on the X chromosome of Drosophila by excision of the w^hd P-element. To investigate the role of nonhomologous end joining (NHEJ) and homologous recombination (HR) in the repair of these breaks, the w^hd P-element was mobilized in flies carrying mutant alleles of either lig4 or rad54. The survival of both lig4- and rad54-deficient males was reduced to 25% in comparison to the wild type, indicating that both NHEJ and HR are involved in the repair P-induced gaps in males. Survival of lig4-deficient females is not affected at all, implying that HR using the homologous chromosome as a template can partially compensate for the impaired NHEJ pathway. In rad54 mutant females survival was reduced to 70% after w^hd excision. PCR analysis indicates that the undamaged homologous chromosome may compensate for the potential loss of the broken chromosome in rad54 mutant females after excision. Molecular analysis of the repair junctions revealed microhomology (2-8 bp)-dependent DSB repair in most products. In the absence of Lig4, the 8-bp target site duplication is used more frequently for repair. These data indicate the presence of efficient alternative end-joining mechanisms, which partly depend on the presence of microhomology but do not require Lig4 (Romeijn, 2005).

Much of the current knowledge of DSB repair in eukaryotes is obtained from studies utilizing rare cutting endonucleases to generate site-specific DSBs in the genome. In Drosophila, P-element transposition has been widely employed as a system to generate site-specific chromosomal breaks. In this study, excision of the 629-bp w^hd P-element was used to induce two staggered DSBs with identical 17-nucleotide 3' overhangs at the site of integration in exon 6 of the white gene. Repair of these breaks is achieved through NHEJ and HR, the two main DSB-repair pathways in eukaryotes. Since the P-element-specific endonuclease is constitutively expressed during development, the P-element is a target to continuous excision. The formation of DSBs is prevented when the breaks are resealed incorrectly via end joining or when in females the homologous chromosome, which does not contain the w^hd P-element, is used as a template for repair. PCR analysis did not reveal the presence of the 629-bp w^hd P-elements in somatic cells of flies containing the Delta2-3 endonuclease. This observation indicates efficient formation of DSBs, resulting in a prevalence of cells in adult flies in which breakage is not possible anymore as a consequence of repair. In repair-proficient flies, the presence of the Delta2-3 element does not influence survival, implying efficient repair of DSBs. P-element excision in F₁ males deficient for lig4 resulted in a 75% reduction in survival. In contrast, the survival of lig4-deficient females was not affected, indicating that allelic recombination can partially compensate for the absence of NHEJ in repair of P-induced gaps. All lig4-deficient females recovered had almost completely wild-type colored eyes. In lig4-proficient females only 30%-40% of each eye was covered with spots. These observations provide direct evidence that repair by HR using the homologous chromosome as a template can effectively compensate for the lack of NHEJ in females in an error-free manner. The drastic reduction in male survival indicates that NHEJ is an essential repair pathway in the absence of a homologous chromosome (Romeijn, 2005).

To determine the role of Lig4 in the repair of breaks after w^hd excision in premeiotic germ cells, male progeny was analyzed by PCR. In males recovered after crossing wild-type and lig4 mutant males the original P-element was found in ~70% of the flies, irrespective of the repair deficiency. In males obtained after crossing both wild-type and lig4 mutant females >95% of the flies still contain the w^hd element. These findings suggest efficient repair through HR using the sister chromatid as a template. Nevertheless, the possibility of reduced endonuclease activity during germ cell formation cannot be excluded (Romeijn, 2005).

P-element excision in rad54 mutant males resulted in a 75% reduction in survival. This strong decrease in survival indicates an important role in males for recombination between sister chromatids in overcoming P-element-induced DSBs. In females, a reduction in survival of ~30% was observed. Remarkably, only 8 of 132 females recovered contained spots of wild-type tissue in the eyes. Apparently, after inactivation of rad54 the homologous chromosome cannot be used anymore as a template for recombination. PCR analysis of females that were recovered showed the presence of the w^a allele in 27 of 36 clones studied. Possibly, breakage of the w^hd chromosome leads to chromosome loss when repair is hampered. Loss of the w^hd chromosome may be tolerated at later stages of development or compensated by duplication of the w^a chromosome. Chromosome loss and duplication has also been observed in certain tumor cells and is one way to explain loss of heterozygosity (Romeijn, 2005).

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).

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date revised: 15 April 2014

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