BIOLOGICAL OVERVIEW

In eukaryotes, genome stability is maintained in part by checkpoint pathways that monitor the state of DNA and regulate the cell division cycle, activate DNA repair, or promote cell death as required. A central place in DNA damage and replication checkpoints in diverse eukaryotes is occupied by homologs of ATM/ATR kinases. Mutations in ATM/ATR homologs have pleiotropic effects that range from sterility to increased killing by genotoxins in humans, mice, and Drosophila. A null allele of mei-41, the Drosophila ATM/ATR homolog has been generated, and it has been used to document a semidominant effect on a larval mitotic checkpoint and methyl methanesulfonate (MMS) sensitivity. The role of mei-41 has been tested in a recently characterized checkpoint that delays metaphase/anaphase transition after DNA damage in cellular embryos. Five existing mei-41 alleles have been tested with respect to known phenotypes (female sterility, cell cycle checkpoints, and MMS resistance). Not all phenotypes are affected equally by each allele, i.e., the functions of Mei-41 in ensuring fertility, cell cycle regulation, and resistance to genotoxins are genetically separable. It is proposed that Mei-41 acts not in a single rigid signal transduction pathway, but in multiple molecular contexts to carry out its many functions. Sequence analysis identified mutations, which, for most alleles, fall in the poorly characterized region outside the kinase domain; this allowed the tentative identification of additional functional domains of Mei-41 that could be subjected to future structure-function studies of this key molecule. The true Drosophila ATM homolog, telomere fusion (common alternative name: ATM), more closely related to ATM, and mei-41 actually belongs to the ATR subfamily (Laurençon, 2003; Song, 2004).

The Schizosaccharomyces pombe homolog, Rad3, was initially identified in a screen for DNA damage sensitive mutants and later found to act in the DNA damage and replication checkpoints (Al-Khodairy, 1992; Seaton, 1992). The Saccharomyces cerevisiae homolog Mec1 was isolated as a gene essential for cell cycle progression (Kato, 1994; Siede, 1996). ATM was identified by studies of the human disease ataxia telangiectasia (AT; Savitsky, 1995a). Additional homologs include AtATM and AtRAD3 of Arabidopsis thaliana, Ce-atl-1 of Caenorhabditis elegans and UVSB of Aspergillus nidulans. All are large proteins of >2500 amino acids that share little similarity outside the kinase domain (Laurençon, 2003 and references therein).

In vitro studies have identified several phosphorylation targets of both ATM and ATR (Kim, 1999). These include other proteins in the checkpoint pathways such as homologs of CHK1 (see Drosophila Grapes) and CHK2 (see Drosophila Loki). These studies have led to a model in which ATM/ATR homologs act early in the checkpoint pathway to sense the presence of damaged or incompletely replicated DNA and relay this signal to the remainder of the checkpoint pathway via phosphorylation. As such, the kinase domain, which is at the C terminus, has been shown to be required for function of both RAD3 and ATR (Jimenez, 1992; Savitsky, 1995a; Savitsky, 1995b; Siede, 1996). For instance, mutations in the catalytic loop of the kinase affect all functions known for RAD3 and ATR, creating dominant negative activities (Bentley, 1996; Cliby, 1998). A temperature-sensitive mutation in the kinase domain of rad3 disrupts the DNA damage checkpoint response and other functions analyzed (Martinho, 1998). Despite its importance, the kinase domain by itself is insufficient for function (Morgan, 1997; Chapman, 1999; Laurençon, 2003 and references therein).

Relatively little, however, is known about the function of sequences outside the kinase domain in the PI3K-l family. In fission yeast, N-terminal sequences consisting of a leucine zipper and a putative protein-protein interaction site called the P site can confer dominant negative activity when overexpressed (Morgan, 1997; Chapman, 1999). A possible explanation for this result is that N-terminal sequences facilitate interaction of PI3K-l proteins with their partners and therefore compete with the endogenous protein when overexpressed (Laurençon, 2003 and references therein).

An ATM/ATR homolog in Drosophila is encoded by mei-41. mei-41 is essential for the DNA damage checkpoint in larval imaginal discs and neuroblasts and for the DNA replication checkpoint in the embryo (Hari, 1995; Brodsky, 2000; Garner, 2001). mei-41 also has an essential role during early nuclear divisions in embryos, where it is required to delay mitosis in response to incomplete DNA replication (Sibon, 1999). Consistent with these functions, mei-41 mutants are sensitive to hydroxyurea, an inhibitor of DNA replication, and DNA-damaging agents such as X-ray and alkylating agents (Boyd, 1976; Sibon, 1999). Mei-41 also plays an important role during meiosis, where it is proposed to monitor double-strand-break repair during meiotic crossing over, to regulate the progression of prophase I, and to enforce metaphase I delay observed at the end of oogenesis (Ghabrial, 1999; McKim, 2000; Laurençon, 2003 and references therein).

All existing mei-41 mutant alleles were isolated in screens for mutants with meiotic defects, female sterility, or increased killing by genotoxins. Therefore, only viable alleles would have been recovered (Baker, 1972; Smith, 1973; Boyd, 1976; Mohler, 1977). One of the strongest of these, mei-41^D3, has been described as a null allele on the basis of the absence of detectable protein and complete female sterility in these mutants (Sibon, 1999). Sequence analysis, however, sheds doubt on whether this allele is a true null that would be a valuable tool for analysis of Mei-41 function (Laurençon, 2003).

A null mutant of mei-41 has now been generated, that is fully viable in the absence of DNA damage and shows a semidominant effect on a larval mitotic checkpoint and methyl methanesulfonate (MMS) sensitivity. This null allele was used to document the role of mei-41 in a recently characterized checkpoint that delays metaphase/anaphase transition in response to DNA damage in cellular embryos (Su, 2001). Five existing mei-41 alleles are compared to the null with respect to known phenotypes (female sterility, G₂/M regulation after DNA damage in larvae, and sensitivity to MMS). Four of these alleles lack meiotic defects; thus, female sterility can be attributed to the failure to regulate syncytial divisions where mei-41 is required to delay mitosis in response to incomplete DNA replication. Interestingly, it was found that not all phenotypes are affected equally by each allele; thus some are separation-of-function alleles. Sequence analysis has identified mutations that reveal the importance of N-terminal sequences and identified putative functional domains of Mei-41. These results support a model in which mei-41 interacts with different sets of upstream and downstream effectors to carry out its many functions (Laurençon, 2003).

ATR homologs act to stall mitosis in response to two types of DNA defects, namely, incompletely replicated DNA and damaged DNA. In Drosophila, mei-41 is required to stall mitosis when DNA replication is blocked experimentally during embryonic cleavage divisions (Sibon, 1999), which occur in a syncytium and are driven by maternally supplied gene products. Embryos from homozygous mothers of strong mei-41 alleles such as D3 and 29D do not progress beyond syncytial cycles (Sibon, 1999). Therefore, in the absence of meiotic defects, female sterility may be attributed to the failure of syncytial divisions. Syncytial division defects have been proposed (Sibon, 1999) to occur due to a failure to delay mitosis in the presence of ongoing DNA replication during these rapid division cycles (Laurençon, 2003).

According to the above discussion, mei-41^D12 and mei-41^D13 mutants, which present a wild-type level of syncytial division function (because they are female fertile) but are unable to regulate metaphase/anaphase in cellularized embryos or G₂/M transition in embryos and larvae following DNA damage, may be responsive to incompletely replicated DNA replication but not to damaged DNA. This could be because syncytial divisions simply require less Mei-41 activity than do larval and embryonic checkpoints. Indeed, 29D heterozygotes, which presumably have wild-type Mei-41 but at reduced levels, have normal female fertility but an ~50% loss of larval G₂/M checkpoint. If so, however, it would be expected that mei-41^D14 mutants, which show a more severe fertility phenotype, would be more defective than mei-41^D12 or mei-41^D13 mutants for larval mitotic checkpoint. This is not the case. Therefore, it is proposed, instead, that mei-41^D12 and mei-41^D13 represent separation-of-function alleles that retain normal activity for syncytial divisions but not for DNA damage checkpoints. Conversely, mei-41^D14 allele is compromised for syncytial cycle function while retaining nearly wild-type activity for larval mitotic checkpoints. Such alleles should be potentially useful for identifying genes that interact with mei-41 in one context but not another. Additionally, all five alleles (i.e., all except D3 and D5) show a wild-type level of meiotic function as previously described and yet show defects in mitotic cycles during embryogenesis and larval development. Thus, all represent separation-of-function alleles that have normal meiotic function but are defective for regulation of mitotic proliferation (Laurençon, 2003).

Interestingly, mei-41^D15 mutants that have a wild-type level of mitotic checkpoint are more MMS sensitive than mei-41^D12 mutants that have a severely defective mitotic checkpoint. Likewise, mei-41^D14 and mei-41^D15 mutants show significantly different levels of female fertility (and thus, possibly different DNA replication checkpoint activity), yet have similar MMS sensitivity. It is proposed that defects in cell cycle regulation cannot fully explain the MMS sensitivity of mei-41 mutants. It is likely that the role of mei-41 in DNA repair, cell death, and other yet-to-be-characterized processes contribute to MMS sensitivity (Laurençon, 2003).

One key question concerning checkpoint proteins is whether they are essential for viability in unperturbed cell cycles or they are essential only in the presence of genetic aberrations. Some PI3K-l proteins are essential for cellular viability, while others are not. mec1 deletion mutants of budding yeast are inviable and ATR mutant mice die after the blastocyst stage (Kato, 1994; Brown, 2000). However, the S. pombe rad3Delta strain, in which rad3 is disrupted, is viable and the ATM mutations observed to date in mice are fully viable (Jimenez, 1992; Seaton, 1992; Barlow, 1996; Elson, 1996; Xu, 1996). Null mutants of mei-41 are viable, since homozygous mutant flies are produced from heterozygous parents. It is possible, however, that homozygous mei-41 mutant progeny survive due to a supply of wild-type Mei-41 deposited into the eggs by heterozygous mothers. Because embryos from homozygous mutant females fail to progress beyond cleavage divisions (Sibon, 1999), mei-41 does have an essential role in early embryogenesis (Laurençon, 2003).

Interestingly, heterozygotes for the null allele of mei-41 show checkpoint defects and MMS sensitivity. Heterozygous phenotypes have been described for ATM-deficient cells of human and mouse, including increased sensitivity to killing by mutagens, defective cell cycle checkpoints, and chromosome aberrations, among others (Naeim, 1994; Scott, 1994; Tchirkov, 1997; Djuzenova, 1999). In mice, heterozygous ATR ES cells do not display increased sensitivity to DNA-damaging agents although other phenotypes such as cell cycle regulation remain to be assayed (De Klein, 2000). Haplo-insufficiency of ATM/ATR homologs for checkpoint regulation may explain why heterozygous mutant mice display increased tumor incidence (Brown, 2000). This notion remains controversial, however, because recent work suggests that the ATM heterozygotes in question may harbor a mutant allele that acts in a dominant negative manner to inhibit the remaining wild-type allele (Spring, 2002). The null allele in Drosophila, mei-41^29D, is predicted to encode only 39 aa and is therefore unlikely to produce a dominant negative Mei-41. As such, Mei-41 may be truly haplo-insufficient for optimal checkpoint regulation (Laurençon, 2003).

This study documents a novel role for mei-41 in the regulation of metazoan mitotic progress. DNA damage blocks mitosis but the exact mitotic step blocked can differ from cell type to cell type (Elledge, 1996). In fission yeast, the entry into mitosis is blocked whereas in budding yeast, chromosome segregation and metaphase/anaphase transition are blocked. In contrast, Drosophila and human cells appear capable of blocking both the entry into mitosis and the metaphase/anaphase transition (Smits, 2000; Su, 2000; Su, 2001). The role of mei-41 in blocking the entry into mitosis after DNA damage has been documented previously, but this report documents for the first time that an ATM/ATR homolog is needed to block mitotic progression in metazoa. Moreover, the results rule out the possibility that damaged chromosomes present a physical barrier to chromosome separation and consequently delay anaphase. Rather, the delay of anaphase is more likely to be an active response since it requires a checkpoint gene (Laurençon, 2003).

This study describes several mutations that fall outside of the kinase domain but appear to affect mei-41 function profoundly. Although the unique mutations described are most likely culprits for the phenotype of each allele, the contribution of other mutations present within or without the mei-41 coding region cannot be ruled out. Site-directed mutagenesis to obliterate domains implicated by these data would be valuable in addressing this issue unequivocally. Nonetheless, in fission yeast, N terminus of RAD3 and, in particular, a putative leucine zipper and a putative protein-protein interaction domain can confer dominant functions when overexpressed (Chapman, 1999). In humans, N-terminal 247 aa of ATM are required for interaction with p53 in vitro (Khanna, 1998). Thus, N-terminal sequences may contribute to the function of ATM/ATR family members via protein-protein interaction. Interestingly, in two-hybrid assays, a Mei-41 N-terminal fragment containing this helix interacts with MUS304, a protein needed for larval DNA damage checkpoint (Brodsky, 2000) and a homolog of mammalian ATRIP proteins (Laurençon, 2003).

In conclusion, this study demonstrates that mei-41 null mutants are viable and show dose-sensitive defects in cell cycle checkpoints and MMS sensitivity. Sequence analysis reveals the importance of the kinase domain in all aspects of Mei-41 function and identifies a putative alpha-helix in the N terminus, which may be important for mei-41 function in DNA-damage-induced G₂/M checkpoint and syncytial divisions. Two other mutations located in a putative helix-loop-helix in the N terminus and a conserved amino acid in the rad3/FAT domain may be causing defective DNA-damage checkpoint while sparing syncytial division functions. These possibly represent separation-of-function alleles of mei-41, which may be useful in screens for interacting genes. The fact that not all phenotypes are affected equally by the five alleles studied is consistent with the idea that mei-41 operates in many different molecular contexts to carry out its many functions. There is precedent for this idea because grapes, a Drosophila chk1 homolog, that is thought to function downstream of mei-41, appears to do so in regulation of mitosis but not of meiosis. The data suggest that even in mitotic cycles, signaling networks in which mei-41 participates may not be rigid, but change at different stages in development (syncytial vs. larval) or in response to different kinds of DNA defects (DNA damage vs. incomplete replication) (Laurençon, 2003).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Nuclear lamina dysfunction triggers a germline stem cell checkpoint

LEM domain (LEM-D) proteins are conserved components of the nuclear lamina (NL) that contribute to stem cell maintenance through poorly understood mechanisms. The Drosophila emerin homolog Otefin (Ote) is required for maintenance of germline stem cells (GSCs) and gametogenesis. This study shows that ote mutants carry germ cell-specific changes in nuclear architecture that are linked to GSC loss. Strikingly, both GSC death and gametogenesis are rescued by inactivation of the DNA damage response (DDR) kinases, ATR and Chk2. Whereas the germline checkpoint draws from components of the DDR pathway, genetic and cytological features of the GSC checkpoint differ from the canonical pathway. Instead, structural deformation of the NL correlates with checkpoint activation. Despite remarkably normal oogenesis, rescued oocytes do not support embryogenesis. Taken together, these data suggest that NL dysfunction caused by Otefin loss triggers a GSC-specific checkpoint that contributes to maintenance of gamete quality (Barton, 2018).

The Drosophila emerin homolog Ote has an essential requirement for GSC survival and germ cell differentiation. This study shows that Ote loss causes GSC-specific nuclear defects that include a thickened and irregular NL and aggregation of heterochromatin. Strikingly, inactivation of two DDR kinases, ATR, and Chk2, rescues oogenesis in ote-/- females, a rescue that is cell-type specific. Genetic and cytological features of the checkpoint pathway present in ote mutant GSCs differ from those found in canonical DDR pathways. In addition, although heterochromatin coalesce is present, such defects by themselves do not trigger the checkpoint. Instead, the data correlate Chk2 activity with defects in NL structure, indicating that NL dysfunction is responsible for the activation of a checkpoint pathway in GSCs. Despite remarkably normal oogenesis, rescued oocytes do not support embryogenesis. It is suggested that this NL checkpoint pathway functions in GSCs to ensure that only healthy gametes are passed on to the next generation (Barton, 2018).

These studies identify ATR as the critical responder kinase and Chk2 as the critical transducer kinase in the NL checkpoint. This signaling axis differs from the canonical ATM-to-Chk2 or ATR-to-Chk1 axes. Several factors might contribute to the choice of responder and transducer kinase. First, species-specific constraints might exist. ATR, but not ATM is essential in mammals, whereas ATM, but not ATR, is essential in Drosophila. Second, cell-type specific distinctions are apparent. In both the fly and mouse germline, persistent meiotic double-strand breaks activate ATR and Chk2, implying that the ATR-Chk2 axis might be dominant in germ cells. Third, the nature of the trigger might influence which proteins are involved in signaling. For example in Drosophila, ATR and Chk2 are both required for the patterning defects caused by a failure to repair meiotic double-strand breaks. However, in DNA-damaged GSCs, ATR protects against GSC death, whereas Chk2 promotes it. These data suggest that in the case of the NL checkpoint, both ATR and Chk2 promote germ cell death. These studies add to growing evidence that the DDR pathway is modular, with selective use of pathway components in response to various cellular stresses (Barton, 2018).

Activated Chk2 is commonly associated with phosphorylation and activation of p53. Canonically, p53 activation leads to cell cycle arrest and apoptosis. These studies demonstrate that GSC loss persists in ote-/-; p53-/- females, suggesting that classical apoptosis is not responsible for GSC death. These findings are consistent with the absence of classic markers of apoptosis in ote mutants and observations that the p53 regulatory network differs in GSCs. Recently, an alternative cell death pathway was identified in spermatogonia of Drosophila testes. This pathway is responsible for spontaneous elimination of spermatogonia, using activated lysosomal and mitochondrial-associated factors. Additional studies are needed to determine whether ATR/Chk2-dependent GSC loss in ote mutants targets a similar pathway (Barton, 2018).

The data suggest that NL dysfunction is the primary cause of the ATR/Chk2 checkpoint in GSCs. Notably, NL defects are found only in affected cells and persist in rescued chk2-/-, ote-/- double mutants. Multiple mechanisms might connect nuclear architecture changes to ATR/Chk2 activation. First, altered NL structure might change genomic contacts needed for appropriate transcriptional regulation, with resulting gene expression changes prompting activation of the checkpoint. While global transcriptional changes during oogenesis were not observed, identification of transcriptional changes specific to GSCs or early germ cells would have been masked in these studies. Second, disruptions in the NL might affect trafficking of products between the nucleus and cytoplasm. Notably, a recent study identified large ribonucleoparticles (megaRNPs) that exit the nucleus by egress or budding through the inner and outer nuclear membranes, a process disrupted by defects in the NL. As such, it remains possible that the thickened NL in ote-/- GSCs disrupts large ribonucleoprotein (megaRNP) egress, leading to cellular stress and ATR/Chk2 activation. Third, defects in the NL structure might alter scaffolding of components of the DDR pathway, leading to checkpoint activation. Indeed, proteomic studies from Drosophila cultured somatic cells found that Ote interacted with proteins involved in DNA replication and repair, implying that Ote might assemble responder and transducer kinases complexes at the NL. However, observations that the ATR/Chk2-dependent checkpoint is GSC-specific, coupled with findings that meiotic double-strand breaks are repaired appropriately in chk2-/-, ote-/- germaria, argue against this model. Fourth, structural alteration in the nuclear envelope itself might trigger ATR/Chk2 activation. Indeed, emerging evidence implicates ATR as a general sensor of the structural integrity of cellular components. Further studies are needed to identify how NL dysfunction triggers the GSC-specific checkpoint (Barton, 2018).

Mutations in NL LEM-D proteins cause dystrophic diseases. Much evidence suggests that these diseases result from compromised stem cell populations that underlie the defects in tissue homeostasis. Indeed, a wealth of evidence links NL defects to increased DNA damage. The data are consistent with these reports, as it was shown that elevated accumulation of the commonly used DNA damage marker. However, this study found that phosphorylation of the H2A variant occurs downstream of Chk2, suggesting that accumulation of DNA damage in cells with a dysfunctional NL might be a consequence of cells dying, not the primary cause. These unexpected results suggest that caution is needed in linking causation of γH2Av/H2X accumulation to DNA damage and a failure in DNA repair. Indeed, recent studies of progerin-expressing cells indicated that the cellular defect in Hutchinson-Gilford progeria cells does not lie in defective DNA repair and DNA damage, even though these cells accumulate phosphorylated H2AX. These findings establish a new context for consideration of mechanisms of laminopathic diseases, suggesting that detrimental effects of NL dysfunction are primary events that are linked to checkpoint activation and stem cell loss (Barton, 2018).

PROTEIN STRUCTURE

Amino Acids - 2354

Structural Domains

The predicted mei-41 protein is similar in sequence to the ATM (ataxia telangiectasia) protein from humans and to the yeast rad3 and Mec1p proteins (Hari, 1995).

date revised: 22 February 2022

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