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
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).
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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