Gene name - meiotic 41
Cytological map position - 14C3
Function - enzyme
Symbol Symbol - mei-41
FlyBase ID: FBgn0004367
Genetic map position - 1-54.2
Classification - ATM/ATR kinase, phosphatidylinositol 3-kinase
Cellular location - cytoplasmic
|Recent literature||Park, J. S., Na, H. J., Pyo, J. H., Jeon, H. J., Kim, Y. S. and Yoo, M. A. (2015). Requirement of ATR for maintenance of intestinal stem cells in aging Drosophila. Aging (Albany NY) [Epub ahead of print]. PubMed ID: 26000719
The stem cell genomic stability forms the basis for robust tissue homeostasis, particularly in high-turnover tissues. For the genomic stability, DNA damage response (DDR) is essential. This study focused on the role of two major DDR-related factors, ataxia telangiectasia-mutated (ATM) and ATM- and RAD3-related (ATR) kinases, in the maintenance of intestinal stem cells (ISCs) in the adult Drosophila midgut. ATM and ATR phosphorylate their substrates, including H2AX and p53, preferentially on a serine or threonine preceding a glutamine (pS/TQ). The role of ATM and ATR was explored utilizing immunostaining with an anti-pS/TQ antibody as an indicator of ATM/ATR activation, gamma-irradiation as a DNA damage inducer, and the UAS/GAL4 system for cell type-specific knockdown of ATM, ATR, or both during adulthood. The results showed that the pS/TQ signals got stronger with age and after oxidative stress. The pS/TQ signals were found to be more dependent on ATR rather than on ATM in ISCs/enteroblasts (EBs). Furthermore, an ISC/EB-specific knockdown of ATR, ATM, or both decreased the number of ISCs and oxidative stress-induced ISC proliferation. The phenotypic changes that were caused by the ATR knockdown were more pronounced than those caused by the ATM knockdown; however, the data indicate that ATR and ATM are both needed for ISC maintenance and proliferation; ATR seems to play a bigger role than does ATM.
|Pinto, B. S. and Orr-Weaver, T. L. (2017). Drosophila protein phosphatases 2A B' Wdb and Wrd regulate meiotic centromere localization and function of the MEI-S332 Shugoshin. Proc Natl Acad Sci U S A. PubMed ID: 29158400
Proper segregation of chromosomes in meiosis is essential to prevent miscarriages and birth defects. This requires that sister chromatids maintain cohesion at the centromere as cohesion is released on the chromatid arms when the homologs segregate at anaphase I. The Shugoshin proteins preserve centromere cohesion by protecting the cohesin complex from cleavage, and this has been shown in yeasts to be mediated by recruitment of the protein phosphatase 2A B' (PP2A B'). In metazoans, delineation of the role of PP2A B' in meiosis has been hindered by its myriad of other essential roles. The Drosophila Shugoshin MEI-S332 can bind directly to both of the B' regulatory subunits of PP2A, Wdb and Wrd, in yeast two-hybrid experiments. Exploiting experimental advantages of Drosophila spermatogenesis, this study found that the Wdb subunit localizes first along chromosomes in meiosis I, becoming restricted to the centromere region as MEI-S332 binds. Wdb and MEI-S332 show colocalization at the centromere region until release of sister-chromatid cohesion at the metaphase II/anaphase II transition. MEI-S332 is necessary for Wdb localization, but, additionally, both Wdb and Wrd are required for MEI-S332 localization. Thus, rather than MEI-S332 being hierarchical to PP2A B', these proteins reciprocally ensure centromere localization of the complex. Functional relationships between MEI-S332 and the two forms of PP2A were analyzed by quantifying meiotic chromosome segregation defects in double or triple mutants. These studies revealed that both Wdb and Wrd contribute to MEI-S332's ability to ensure accurate segregation of sister chromatids, but, as in centromere localization, they do not act solely downstream of MEI-S332.
|Brady, M. M., McMahan, S. and Sekelsky, J. (2018). Loss of Drosophila Mei-41/ATR alters meiotic crossover patterning. Genetics 208(2): 579-588. PubMed ID: 29247012
Meiotic crossovers must be properly patterned to ensure accurate disjunction of homologous chromosomes during meiosis I. Disruption of the spatial distribution of crossovers can lead to nondisjunction, aneuploidy, gamete dysfunction, miscarriage, or birth defects. One of the earliest identified genes involved in proper crossover patterning is Drosophila mei-41, which encodes the ortholog of the checkpoint kinase ATR. Analysis of hypomorphic mutants suggested the existence of crossover patterning defects, but it was not possible to assess this in null mutants because of maternal-effect embryonic lethality. To overcome this lethality, mei-41 null mutants were constructed in which wild-type Mei-41 was expressed in the germline after completion of meiotic recombination, allowing progeny to survive. Crossovers were decreased to about one-third of wild-type levels, but the reduction is not uniform, being less severe in the proximal regions of chromosome 2L than in medial or distal 2L or on the X chromosome. None of the crossovers formed in the absence of Mei-41 require Mei-9, the presumptive meiotic resolvase, suggesting that Mei-41 functions everywhere, despite the differential effects on crossover frequency. Interference appears to be significantly reduced or absent in mei-41 mutants, but the reduction in crossover density in centromere-proximal regions is largely intact. It is proposed that crossover patterning is achieved in a stepwise manner, with the crossover suppression related to proximity to the centromere occurring prior to and independently of crossover designation and enforcement of interference. In this model, Mei-41 has an essential function in meiotic recombination after the centromere effect is established but before crossover designation and interference occur.
|Kizhedathu, A., Bagul, A. V. and Guha, A. (2018). Negative regulation of G2-M by ATR (mei-41)/Chk1(Grapes) facilitates tracheoblast growth and tracheal hypertrophy in Drosophila. Elife 7. PubMed ID: 29658881
Imaginal progenitors in Drosophila are known to arrest in G2 during larval stages and proliferate thereafter. This study investigated the mechanism and implications of G2 arrest in progenitors of the adult thoracic tracheal epithelium (tracheoblasts). Tracheoblasts were shown to pause in G2 for ~48-56 h and grow in size over this period. Surprisingly, tracheoblasts arrested in G2 express drivers of G2-M like Cdc25/String (Stg). Mechanisms that prevent G2-M are also in place in this interval. Tracheoblasts activate Checkpoint Kinase 1/Grapes (Chk1/Grp) in an ATR/mei-41-dependent manner. Loss of ATR/Chk1 led to precocious mitotic entry ~24-32 h earlier. These divisions were apparently normal as there was no evidence of increased DNA damage or cell death. However, induction of precocious mitoses impaired growth of tracheoblasts and the tracheae they comprise. It is proposed that ATR/Chk1 negatively regulate G2-M in developing tracheoblasts and that G2 arrest facilitates cellular and hypertrophic organ growth.
|Bayer, F. E., Zimmermann, M., Preiss, A. and Nagel, A. C. (2018). Overexpression of the Drosophila ATR homologous checkpoint kinase Mei-41 induces a G2/M checkpoint in Drosophila imaginal tissue. Hereditas 155: 27. PubMed ID: 30202398
DNA damage generally results in the activation of ATM/ATR kinases and the downstream checkpoint kinases Chk1/Chk2. In Drosophila melanogaster, the ATR homologue meiotic 41 (mei-41) is pivotal to DNA damage repair and cell cycle checkpoint signalling. Although various mei-41 mutant alleles have been analyzed in the past, no gain-of-function allele is yet available. To fill this gap, transgenic flies were generated allowing temporal and tissue-specific induction of mei-41. Overexpression of mei-41 in wing and eye anlagen affects proliferation and a G2/M checkpoint even in the absence of genomic stress. Similar consequences were observed following the overexpression of the downstream kinase Grapes (Grp) but not of Loki (Lok), encoding the respective Drosophila Chk1 and Chk2 homologues, in agreement with their previously reported activities. Moreover, this study showed that irradiation induced cell cycle arrest was prolonged in the presence of ectopic mei-41 expression. Similar to irradiation stress, mei-41 triggered the occurrence of a slower migrating form of Grp, implying specific phosphorylation of Grp in response to either signal. Using a p53R-GFP biosensor, it was further shown that overexpression of mei-41 was sufficient to elicit a robust p53 activation in vivo. It is concluded that overexpression of the Drosophila ATR homologue mei-41 elicits an effectual DNA damage response irrespective of irradiation.
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-41D3, 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-41D12 and mei-41D13 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-41D14 mutants, which show a more severe fertility phenotype, would be more defective than mei-41D12 or mei-41D13 mutants for larval mitotic checkpoint. This is not the case. Therefore, it is proposed, instead, that mei-41D12 and mei-41D13 represent separation-of-function alleles that retain normal activity for syncytial divisions but not for DNA damage checkpoints. Conversely, mei-41D14 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-41D15 mutants that have a wild-type level of mitotic checkpoint are more MMS sensitive than mei-41D12 mutants that have a severely defective mitotic checkpoint. Likewise, mei-41D14 and mei-41D15 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-4129D, 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).
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-41D3;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-41D3 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-41D3;tefu8 double mutant germaria were examined. At a permissive temperature (18°), mei-41D3;tefu8 displayed a γ-H2AV staining pattern similar in severity to mei-41D3 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-41D3;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-41D3;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-41D3;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 MRG15j6A3 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-41D3, 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-41D3, 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).
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: 5 July 2003
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