Six mutant alleles of the mei-41 locus in Drosophila have been shown to cause hypersensitivity to hydroxyurea in larvae. The strength of that sensitivity is directly correlated with the influence of the mutant alleles on meiosis in that: alleles exhibiting a strong meiotic effect (mei-41D2, mei-41D5, mei-41D7) are highly sensitive; alleles with negligible meiotic effects (mei-41(104)D1, mei-41(104)D2) are moderately sensitive and an allele which expresses meiotic effects only under restricted conditions (mei-41D9) has an intermediate sensitivity. This sensitivity is not a general feature of strong postreplication repair-deficient mutants, because mutants with that phenotype from other loci do not exhibit sensitivity (mus(2)205A1, mus(3)302D1, mus(3)310D1). The observed lethality is not due to hypersensitivity of DNA synthesis in mei-41 larvae to hydroxyurea as assayed by tritiated thymidine incorporation. Lethality is, however, potentially attributable to an abnormal enhancement of chromosomal aberrations by hydroxyurea in mutant mei-41 larvae. Both in vivo and in vitro exposure of neuroblast cells to hydroxyurea result in an increase in 3 types of aberrations that is several fold higher in mei-41 tissue. Since hydroxyurea disrupts DNA synthesis, these results further implicate the mei-41 locus in DNA metabolism and provide an additional tool for an elucidation of its function. The possible existence of additional genes of this nature is suggested by a more modest sensitivity to hydroxyurea which has been detected in two stocks carrying mutagen-sensitive alleles of alternate genes (Banga, 1986).
The mei-41 gene of Drosophila plays an essential role in meiosis, in the maintenance of somatic chromosome stability, in postreplication repair and in DNA double-strand break repair. This gene has been cytogenetically localized to polytene chromosome bands 14C4-6 using available chromosomal aberrations. About 60 kb of DNA sequence has been isolated following a bidirectional chromosomal walk that extends over the cytogenetic interval 14C1-6. The breakpoints of chromosomal aberrations identified within that walk establish that the entire mei-41 gene has been cloned. Two independently derived mei-41 mutants have been shown to carry P insertions within a single 2.2 kb fragment of the walk. Since revertants of those mutants have lost the P element sequences, an essential region of the mei-41 gene is present in that fragment. A 10.5 kb genomic fragment that spans the P insertion sites has been found to restore methyl methanesulfonate resistance and female fertility of the mei-41D3 mutants. The results demonstrate that all the sequences required for the proper expression of the mei-41 gene are present on this genomic fragment. This study provides the foundation for molecular analysis of a function that is essential for chromosome stability in both the germline and somatic cells (Banga, 1995).
The D. melanogaster mei-41 gene is required for DNA repair, mitotic chromosome stability, and normal levels of meiotic recombination in oocytes. The predicted mei-41 protein is similar in sequence to the ATM protein from humans and to the yeast rad3 and Mec1p proteins. There is also extensive functional overlap between mei-41 and ATM. Like ATM-deficient cells, mei-41 cells are exquisitely sensitive to ionizing radiation and display high levels of mitotic chromosome instability. Mei-41 cells, like ATM-deficient cells, fail to show an irradiation-induced delay in the entry into mitosis that is characteristic of normal cells. Thus, the mei-41 gene of Drosophila may be considered to be a functional homolog of the human ATM gene (Hari, 1995).
The phosphoinositide 3-kinase-related kinases (PIKK) family in mammals includes six subfamilies based on sequence homology and function. Human ataxia-telangiectasia mutated (ATM) and ATM and Rad-3-related (ATR) proteins are the key kinases that transduce signals in response to various types of DNA damage. Drosophila mei-41 was originally reported as the ATM ortholog. However, sequence analysis reveals that CG6535 [see telomere fusion (common alternative name: ATM)], a gene predicted by the annotated Drosophila genome, is more closely related to ATM, and mei-41 actually belongs to the ATR subfamily. A related kinase ATX (CG32743 in Drosophila) plays a role in eliminating RNA species containing premature termination codons in C. elegans, whereas in humans it may have a role in the DNA damage response. Two other PIKK family members Drosophila CG2905 and CG5092 are closely related to TRRAP and mTOR, respectively, whereas the catalytic subunit of DNA-dependent protein kinase is not present in Drosophila. Among these kinases, flies with mutations in mei-41 and mTOR have been studied in detail, whereas the functions of CG2905, CG6535, and CG32743 in Drosophila remain unknown (Song, 2004 and references therein).
mei-41 is required during meiosis for the precocious anaphase observed in crossover-defective mutants. Normally in Drosophila oocytes, tension on the meiotic spindle causes a metaphase I arrest. This tension results because crossovers, and the resulting chiasmata, hold homologs together that are being pulled by kinetochore microtobules toward opposite spindle poles. In the absence of tension, such as in a recombination-defective mutant, metaphase arrest is not observed and meiosis proceeds through the two divisions. In some recombination-defective mutants, the precocious anaphase requires the mei-41 gene product. For example, metaphase arrest is not observed in mei-218 mutants because of the severe reduction in crossing over. In mei-41;mei-218 double mutants, however, metaphase arrest is restored. The effect of mei-41 is dependent on double-strand break formation. Thus, in mutants that fail to initiate meiotic recombination the absence of mei-41 has no effect (McKim, 2000).
The genes okra and spindle-B act during meiosis in Drosophila to repair double-stranded DNA breaks (DSBs) associated with meiotic recombination. spn-B (DMC1/RAD51-like) and okr (Dmrad54) are homologous to genes in the yeast RAD52 epistasis group that function in the recombinational repair of DSBs; in Drosophila, mutations in these genes lead to mitotic and/or meiotic defects, consistent with a requirement in DNA repair. RAD51, which promotes homologous pairing and strand exchange during double strand break repair, is a single-stranded DNA binding protein and possesses DNA-stimulated ATPase activity. Unexpectedly, mutations in spn-B and okr cause dorsoventral patterning defects during oogenesis. These defects result from a failure to accumulate Gurken protein, which is required to initiate dorsoventral patterning during oogenesis. The block in Gurken accumulation in the oocyte cytoplasm reflects activation of a meiotic checkpoint in response to the persistence of DSBs in the nucleus. Vasa is a target of this meiotic checkpoint, and so may mediate the checkpoint-dependent translational regulation of Gurken (Ghabrial, 1999).
One of the checkpoint genes required to arrest meiosis in response to the presence of unrepaired DSBs is MEC1, which encodes a member of the ATM/ATR subfamily of phosphatidylinositol-3-OH-kinase-like proteins. In otherwise wild-type yeast, mec1 mutations lead to occasional premature meiosis I, as indicated by the persistence of foci of Rad51 (believed to represent sites of DSB repair) on metaphase-I chromosomes. These results indicate that Mec1 may normally act to delay the cell cycle in the presence of repair intermediates. In Drosophila, mei-41 encodes a homolog of Mec1, and mei-41 mutants show meiotic non-disjunction as well as maternal-effect defects in the timing of mitotic cell cycles in the early embryo (Ghabrial, 1999).
To test whether the production of patterning defects by mutations in the spindle-class genes is due to the engagement of an analogous meiotic checkpoint, flies were made doubly mutant for okr, spn-B or spn-C and mei-41. Mutations in mei-41 are indeed able to suppress the dorsoventral patterning defects caused by mutations in the spindle-class genes. In double-mutant flies, a dramatic increase is observed in the accumulation of Grk protein, as indicated by whole-mount antibody staining and by restoration of anteroposterior and dorsoventral patterning in the eggshell. Significant suppression of the oocyte nuclear-morphology defect is also observed. Suppression of the spindle-class defects by mei-41 is not as complete as that by mei-W68, raising the possibility that there may be some functional redundancy between mei-41 and a putative Drosophila ATM homolog, as appears\ to be the case for yeast MEC1 and TEL1. From these results, it is concluded that the patterning defects observed in mutants of the spindle class are caused by the activation of a mei-41-dependent checkpoint pathway in response to the persistence of unrepaired DSBs during meiosis (Ghabrial, 1999).
Vasa is a target of the meiotic checkpoint. These results raise the question of how the mei-41-dependent checkpoint pathway affects accumulation of Grk. One candidate for a downstream target and effector of the mei-41-dependent pathway is the product of the vasa (vas) gene. vas encodes a protein similar to the translation-initiation factor eIF4A, produces mutant phenotypes similar to those observed in okr, spn-B and spn-C mutants, and has been implicated in the translational control of Grk and certain other oocyte-specific proteins. However, unlike okr, spn-B and spn-C mutations, mutations in vas are not suppressed by mutations in mei-41. The karyosome phenotype of vas mutants is also not suppressed by mei-41 mutations. This difference between vas and the spindle-class mutants indicates that Vas may act downstream of this mei-41-dependent meiotic checkpoint. To address this question more directly, Vas expression was studied in spindle-class-mutant backgrounds. At the level of whole-mount antibody staining, Vas does not appear to be affected. However, the mobility of Vas, as assessed by SDS polyacrylamide gel electrophoresis (SDS-PAGE), is altered: Vas migration appears to be retarded in spn-B mutant ovaries as compared with wild-type ovaries or ovaries heterozygous for spn-B. These results indicate that Vas might be post-translationally regulated by the mei-41-dependent checkpoint pathway. In support of this interpretation, the mobility of Vas from ovarian lysates prepared from flies doubly mutant for spn-B and mei-W68 or mei-41 is restored to that observed in wild-type lysates. Taken together, these data support a model in which activation of the mei-41-dependent checkpoint pathway occurs in response to the presence of DSBs and leads to the modification of Vas, resulting in the downregulation of its activity and a consequent decrease in Grk translation (Ghabrial, 1999).
In yeast, activation of the recombination checkpoint downregulates transcription of genes that are targets of transcription factor Ndt80, including cyclins required for progression through meiosis and a set of sporulation genes required for the morphological changes that normally accompany yeast meiosis. It is proposed that, in Drosophila, activation of this checkpoint pathway results in the modification of Vas and downregulation of the translation of Vas targets such as Grk. In this regard, phosphorylation has been suggested to be a mechanism for downregulating the activity of several translational activators, including Vas-like proteins, plant eIF4A and vertebrate p68 RNA helicase (Ghabrial, 1999 and references therein).
Drosophila embryogenesis is initiated by 13 rapid syncytial mitotic divisions that do not require zygotic gene activity. This maternally directed cleavage phase of development terminates at the midblastula transition (MBT), at which point the cell cycle slows dramatically, membranes surround the cortical nuclei to form a cellular blastoderm, and zygotic gene expression is first required. Embryos lacking Mei-41, a Drosophila homolog of the ataxia telangiectasia (ATM) tumor suppressor, proceed through unusually short syncytial mitoses, fail to terminate syncytial division following mitosis 13, and degenerate without forming cells. Transcription of gap and pair rule genes show expected expression patterns during syncytial divisions 12 and 13, but on completion of mitosis 13 the expected high-level, spatially restricted expression of these genes is not initiated. Mutations in mei-41 thus prevent the onset of the high-level, patterned gene expression that characterizes the transition from maternal to zygotic control of development. It is thought that mei-41 has a specific role in zygotic gene activation (Sibon, 1999).
A similar cleavage-stage arrest is produced by mutations in grapes, which encodes a homolog of the Checkpoint-1 kinase. Biochemical, cytological and genetic data is presented indicating that Mei-41 and Grapes are components of a conserved DNA-replication/damage checkpoint pathway that triggers inhibitory phosphorylation of the Cdc2 kinase and mediates resistance to replication inhibitors and DNA-damaging agents. Double grapes:mei-41 mutants are indistinguishable from either single mutant, supporting the hypothesis that the two genes function in the same pathway. The grp null allele is a dominant enhancer of the mei-41 embryonic lethality, further supporting the hypothesis that Mei-41 and Grapes function in the same pathway during embryogenesis. The pathway utilizing Mei-41 and Grape is required to terminate the cleavage stage at the MBT. This pathway is nonessential during postembryonic development. Animals homozygous for null alleles of mei-41 and grp develop to the adult stage, are female-sterile, and produce embryos with normal external morphology that are arrested in the cleavage stage of embryogenesis. Cyclins are required for Cdc2 kinase activity, and mutations in cyclin A and cyclin B bypass the requirement for mei-41 at the MBT. Reduced cyclin A and cyclin B are likely to increase the cell cycle to an extent that allows production of an early zygotic factor that might be required at the MBT. Cyclin mutations do not restore wild-type syncytial cell-cycle timing or the embryonic replication checkpoint, however, suggesting that Mei-41-mediated inhibition of Cdc2 has an additional essential function at the MBT (Sibon, 1999).
The timing of the MBT is controlled by the nucleocytoplasmic ratio, suggesting that this transition, triggered as a result of the titration of a limiting factor by DNA or chromatin during the later cleavage divisions. On the basis of observations made in this study, a model is favored in which a maternal component of the DNA replication machinery serves as the titrated maternal factor that regulates the timing of the MBT. In this model, the replication machinery is in excess and the time required to complete the synthesis (S) phase of the cell cycle is constant until division 10, when at least one replication factor becomes limiting. After this point, the length of time required for chromosome replication progressively increases, and the checkpoint pathway is therefore required to delay mitosis to allow S-phase completion. This model explains the progressive increase in S-phase length during the syncytial-blastoderm divisions, and the requirement for repliction-checkpoint function for these increases. It is concluded that the Drosophila DNA-replication/damage checkpoint pathway can be activated by externally triggered DNA damage or replication defects throughout the life cycle, and under laboratory conditions this inducible function is nonessential for growth to adulthood. During early embryogenesis, however, this pathway is activated by developmental cues and is required for the transition from maternal to zygotic control of development at the MBT (Sibon, 1999).
During early embryogenesis of Drosophila, mutations in the DNA-replication checkpoint lead to chromosome-segregation failures. These segregation failures are associated with the assembly of an anastral microtubule spindle, a mitosis-specific loss of centrosome function, and dissociation of several components of the gamma-tubulin ring complex from a core centrosomal structure. The DNA-replication inhibitor aphidicolin and DNA-damaging agents trigger identical mitotic defects in wild-type embryos, indicating that centrosome inactivation is a checkpoint-independent and mitosis-specific response to damaged or incompletely replicated DNA. It is proposed that centrosome inactivation is part of a damage-control system that blocks chromosome segregation when replication/damage checkpoint control fails (Sibon, 2000).
It has been proposed that a component of the DNA-synthesis machinery becomes rate-limiting during the later syncytial divisions, and that activation of the DNA-replication checkpoint thus produces the increases in embryonic cell-cycle length that characterize these divisions. This model predicts that checkpoint-mutant embryos will initiate mitosis before completing DNA replication specifically during the later syncytial divisions, when the mitotic defects first appear. It was therefore speculated that the centrosome defects and segregation failures in checkpoint-mutant embryos resulted from incomplete DNA replication at mitosis, and that the checkpoint pathway is not specifically required to maintain centrosome function. In wild-type embryos, the DNA-replication inhibitor aphidicolin triggers cell-cycle delays, but mitosis is eventually initiated before S phase is completed. Thus, to test the hypothesis, mitosis was examined following treatment of wild-type embryos with aphidicolin. Under these conditions, gamma-tubulin, and the gamma-tubulin ring complex components Dgrip84 and Dgrip91 are displaced from a core centrosome structure. Aphidicolin triggers mitosis-specific loss of centrosomal gamma-tubulin, Dgrip84 and Dgrip91 during all of the syncytial divisions, in both wild type and checkpoint mutants. This structural response to aphidicolin is therefore replication-checkpoint-independent and is not specific to the later syncytial divisions. This structural response to aphidicoline occurs in grp and mei-41 mutant embryos, and is therefore replication-checkpoint-independent (Sibon, 2000).
To analyse the effects of aphidicolin on spindle assembly and chromosome segregation directly, embryos were injected with a mixture of aphidicolin, rhodamine-labelled tubulin and the DNA label Oligreen and were examined by time-lapse confocal microscopy. The results showed that aphidicolin leads to loss of tubulin foci at nuclear envelope breakdown (NEB), assembly of anastral spindles, and defects in chromosome congression and segregation. Furthermore, the centrosomal foci are re-established on exit from mitosis. Thus aphidicolin treatment accurately phenocopies the mitotic defects observed in checkpoint mutants, indicating that centrosome inactivation and the associated chromosome-segregation failures in these mutants are probably triggered by the presence of incompletely replicated DNA at mitosis (Sibon, 2000).
To determine whether photodamage to DNA also triggers centrosome inactivation and chromosome-segregation defects, laser illumination was used to induce DNA damage. Bipolar spindles are established shortly after NEB, at the time that microtubules originating at the poles interact with each other and with the mitotic chromosomes, and chromosomes rapidly congress to the metaphase plate. During mitosis 12, anaphase is initiated roughly 4 min after NEB, and chromosome segregation is completed and the nuclear envelope begins to reform ~3 min later. However, when the intensity of laser illumination is increased to 90% of maximum, the centrosomal tubulin foci decrease in intensity at NEB and disorganized microtubule bundles form around the condensed chromosomes. Bipolar spindles with broad poles eventually assemble, but the chromosome arms never fully compact at the metaphase plate and chromosome segregation fails. On intense laser illumination in checkpoint mutants and in wild-type embryos, small tubulin-containing dots are often found near poles of the anastral spindles. These structures do not appear to nucleate microtubules during mitosis, but gain nucleating function on exit from mitosis. These structures thus appear to be centrosomes that are inactivated at NEB and reactivated on exit from mitosis. X-ray treatments and ultraviolet light also trigger mitosis-specific loss of centrosomal gamma-tubulin, anastral spindle assembly, and defects in chromosome congression and segregation. On the basis of these observations, it is concluded that a variety of DNA-damaging agents and replication defects can trigger mitosis-specific centrosome inactivation and mitotic chromosome-segregation defects (Sibon, 2000).
Thus, the chromosome-segregation defects in grp and mei-41 checkpoint-mutant embryos are linked to loss of centrosome function and dissociation of several components of the gamma-tubulin ring complex from a core centrosomal structure. However, this does not reflect a specific requirement for grp or mei-41 in maintaining centrosome function. The alleles analysed here are null, yet the mutant embryos proceed through 11-12 normal mitotic divisions before mitotic defects are observed. In addition, replication inhibitors or DNA-damaging agents trigger cytologically identical centrosome defects in wild-type embryos. It is therefore concluded that the loss of centrosome function and mitotic failures in grp and mei-41 mutants reflect activation of a replication-checkpoint-independent response to incomplete DNA replication or DNA damage at mitosis (Sibon, 2000).
Incomplete DNA replication or DNA damage could lead to kinetochore defects, which trigger the spindle-assembly checkpoint; centrosome inactivation could be a response to activation of this checkpoint. However, the localization of gamma-tubulin to the centrosome is not significantly affected by treatment with colchicine, which triggers metaphase arrest through the spindle-assembly checkpoint pathway. In addition, centrosome inactivation does not block centromere alignment, indicating that kinetochore function is not severely compromised when the centrosomes are inactivated. Therefore, DNA-replication- and DNA-damage-associated centrosome inactivation are both independent of the replication and the spindle checkpoints, and appear to reflect the action of a new pathway that links centrosome function to the physical state of the genome (Sibon, 2000).
Mitotic spindle assembly is normally dominated by astral microtubules, which are nucleated at centrosomes. The chromosome-segregation defects in checkpoint mutants and in wild-type embryos treated with aphidicolin or DNA-damaging agents are tightly linked to loss of centrosome-organized microtubules. These observations indicate that asters may be required for spindle function. However, anastral spindles are common during female meiosis and are found in some unusual mitotic systems and cell-free extracts. In addition, spindles in Drosophila embryos mutant for cnn have severely reduced asters, and these anastral spindles may mediate chromosome segregation. Therefore, DNA replication or damage could induce centrosome inactivation and modifications to other functions that are essential for anastral spindle function (Sibon, 2000).
A new role for mei-41 in cell cycle control during meiosis is described. This function is revealed by the requirement of mei-41 for the precocious anaphase observed in crossover-defective mutants. Normally in Drosophila oocytes, tension on the meiotic spindle causes a metaphase I arrest. This tension results because crossovers, and the resulting chiasmata, hold homologs together that are being pulled by kinetochore microtobules toward opposite spindle poles. In the absence of tension, such as in a recombination-defective mutant, metaphase arrest is not observed and meiosis proceeds through the two divisions. In some recombination-defective mutants, the precocious anaphase requires the mei-41 gene product. For example, metaphase arrest is not observed in mei-218 mutants because of the severe reduction in crossing over. In mei-41;mei-218 double mutants, however, metaphase arrest is restored. The effect of mei-41 is dependent on double-strand break formation. Thus, in mutants that fail to initiate meiotic recombination the absence of mei-41 has no effect (McKim, 2000).
The Drosophila ovary is divided into two regions, the germarium, where mitotic divisions occur that produce the oocyte and accompanying nurse cells, and the vitellarium, where oocyte growth and differentiation occur. In the germarium, stem cells produce cystoblasts, which themselves go through four incomplete cell divisions to form a 16-cell cyst. One of the 16 cells becomes the oocyte, and the rest become nurse cells. Meiotic prophase occurs in the germarium and as the cyst enters the vitellarium, meiosis arrests and the chromosomes compact into the karyosome. The nucleus remains in this state until the oocyte is mature and ready for fertilization. At this time, the cell cycle resumes, the nuclear envelope breaks down and metaphase begins. Once again the cell cycle will arrest, this time awaiting passage through the oviduct and fertilization (McKim, 2000 and references therein).
In the absence of crossing over, metaphase arrest does not occur, resulting in precocious anaphase. Metaphase arrest is caused by tension on the meiotic spindle, rather than a signal produced by the crossover events themselves. No precocious anaphase is observed in mei-41D18 or mei-41D3 mutants. This is surprising because mei-41 mutants are crossover defective. Although strong mutants are sterile (and therefore the severity of the recombination defect is unknown), it is reasonable to suppose, extrapolating from the weak alleles, that the defects in the strong alleles are similar to those of mei-9 or mei-218. In double mutants with mei-41, and either mei-9 or mei-218, only oocytes in metaphase arrest are observed. These epistasis results show that the absence of precocious anaphase results from an arrest triggered by the absence of mei-41, rather than there being sufficient crossing over to result in at least one chiasma per nucleus (McKim, 2000).
mei-9 and mei-218 mutants have been shown to have normal levels of gene conversion. These results led to the conclusion that these mutants are defective only in the production of crossovers, and that the initiation of recombination is normal. In contrast, genetic studies have shown that mutations in c(3)G, mei-W68 and mei-P22 result in a failure to initiate meiotic recombination. In fact, the mei-W68 gene encodes a Spo11 homolog, a strong candidate for the enzyme that makes the meiotic double-strand break in S. cerevisiae. Precocious anaphase was observed in mei-41;mei-W68 and mei-41;mei-P22 and mei-41;c(3)G double mutants. The only difference from single mutant oocytes (that is, carrying a wild-type allele of mei-41) is that in some of the mei-41;mei-W68 double mutant oocytes disorganized spindle and chromosomes are observed. These results show that the effect of mei-41 mutants on cell cycle progression is dependent on the initiation of meiotic recombination (McKim, 2000).
It is concluded that in mei-41 mutants a recombination intermediate triggers a change in one or more proteins that regulate the metaphase to anaphase transition. The failure to enter anaphase precociously in mei-41 mutants is only a temporary arrest. Cytological analysis of activated oocytes and embryos from mei-41 homozygous mothers shows that the oocytes eventually enter anaphase, probably during passage through the oviduct. This is also when wild-type oocytes are activated to complete meiosis (McKim, 2000).
Another unusual aspect of the mei-41 mutants is a high frequency of chromosome loss. In most recombination- defective mutant females, there is approximately an equal number of eggs produced with either two X chromosomes (diplo-X) or none (nullo-X). In weak mei-41 mutants, however, there is often a higher frequency of nullo-X gametes compared with diplo-X. This effect is not suppressed by the crossover-defective mutants, but is partially suppressed by the recombination initiation mutants. These results parallel the cytological results and are consistent with the idea that in mei-41 mutants the presence of double-strand breaks has consequences for progression through meiosis and the stability of chromosomes (McKim, 2000).
In mitotic cells, one function of mei-41 is to control the cell cycle via the chk1 protein (Grapes) (Sibon, 1999). To determine whether the activity of mei-41 required for precocious anaphase acts through the chk1 protein, meiosis was observed in mei-9; grapes double mutants. The fact that precocious anaphase was observed in these oocytes shows that the pathway during meiosis is distinct from that in mitotic cells -- one that involves mei-41 signaling to the grapes protein. It is suggested that are at least two pathways through which mei-41 signals during meiosis. As described above, the first is in response to unrepaired double-strand breaks, and may signal through the vasa protein to arrest oocyte development. The second, evidence for which is presented here, is a double strand break-dependent control of the metaphase to anaphase transition. This latter function may be related to its role in meiotic recombination, while the former function might represent a more typical DNA repair pathway. In mitotic cells as well, it has been proposed that ATM homologs signal through different pathways at different times of the cell cycle (Martinho, 1998). The cell cycle effects of mei-41 and spnB mutants are distinct. Ghabrial (1998) discovered a class of Drosophila meiotic mutant that causes sterility because a temporary arrest during meiotic prophase disrupts subsequent developmental events such as oocyte polarity determination. These genes encode double-strand break repair proteins such as Rad54 (okra) and another most like the Rad51 family member XRCC3 (spnB). The cell cycle arrest in these mutants is probably due to the presence of unrepaired double-strand breaks. Two lines of evidence suggest that the cell cycle defects in mei-41 and the spnB/okra mutants are different: (1) mei-41 mutants suppress the cell cycle defect in these mutants (Ghabrial and Schupbach 1999); (2) precocious anaphase is observed in spnB mutants. In summary, the effect of spnB mutants is to delay cell cycle progression during prophase. In mei-41 mutants there are two cell cycle events that do not occur: the prophase delay in spnB mutants, and the precocious anaphase observed in crossover-defective mutants. It is not known whether the double-strand breaks that persist in spnB mutants are repaired in time for metaphase I. In some spnB oocytes, threads of chromatin between the separating masses have been observed, suggesting that there might be types of damage in a spnB mutant that are not repaired prior to the completion of meiosis. A function for mei-41 early in the recombination pathway has effects on the metaphase to anaphase transition; mei-41 and its homolog in S. cerevisiae MEC1 are required for the meiotic-arrest phenotype observed in some meiotic recombination mutants (Lydall, 1996; Ghabrial, 1999). In both Drosophila and S. cerevisiae these genes are involved in the repair of double-strand breaks. These data show a second role for mei-41 in meiotic progression. As suggested by Sekelsky, (1998), the initiation of meiotic recombination may induce a signal that inhibits anaphase. Once the double-strand break is repaired, this signal is turned off, in a process that requires the mei-41 gene. In order to explain the eventual occurrence of anaphase in mei-41 mutants, this inhibitory signal must be overridden by the oviduct-activating signal. This model implies that the function of mei-41 is to monitor repair of the double-strand breaks. An alternative to this checkpoint function, however, is that mei-41 may have a direct role in the repair of double- strand breaks, perhaps by the activation of other repair genes such as spnB. Unlike spnB mutants, a pachytene arrest would not be expected because mei-41 is required for this event. The persisting damage in a mei-41 mutant may activate a checkpoint that can inhibit the metaphase to anaphase transition. In this case, the precocious anaphase observed in spnB mutants suggests the double-strand breaks have been repaired and the cell cycle has resumed prior to oocyte maturation. This repair function for mei-41 might also explain the high level of chromosome loss observed in mutants, since broken chromosomes would be lost during the meiotic divisions (McKim, 2000).
wee has an essential maternal function during the nuclear division cycles of embryogenesis and also implicates zygotic wee function in a cell cycle checkpoint that responds to inhibition of DNA replication. The demonstration that wee has a role during the early syncytial nuclear cycles calls into question a previous assumption that inhibitory phosphorylation does not control these cycles. Analyses of the state of phosphorylation during the early cycles had failed to detect inhibitory phosphorylation of Cdk1 prior to cycle 13. Furthermore, because reduction in the gene dose of cyclin A and cyclin B slows the late nuclear cycles, it has been suggested that progress of these cycles is regulated by accumulation of cyclins to a threshold level. The finding that wee is required for completing the nuclear division cycles suggests that inhibitory phosphorylation plays a role in their regulation after all. The failure to detect inhibitory phosphorylation during these cycles can be explained if only a small pool of Cdk1 is subject to this modification. Wee1-type kinases are predominantly nuclear in Drosophila and other organisms and nuclear Wee1 activity is sufficient to block entry into mitosis even in the presence of high cytoplasmic Cdk1 activity. Hence, it is suggested that inhibitory phosphorylation of a small nuclear pool of Cdk1 contributes importantly to the control of the syncytial cycles. The proposal that inhibitory phosphorylation regulates syncytial cycles is an implicit component of a recently proposed model for the mechanism by which mei-41 and grp regulate the progressive lengthening of these cycles. In response to incompletely replicated DNA, the recognized activities of these conserved checkpoint kinases arrest the cell cycle by preventing the removal of inhibitory phosphates from Cdk1. While this model appears to be at odds with the lack of detectable inhibitory phosphorylation of Cdk1 during the syncytial cycles, the findings that Drosophila wee is required for the early nuclear division cycles supports this proposal. Indeed, the apparent parallels in the phenotypes of mei-41, grp, and wee maternal mutants suggest that these genes operate by a similar mechanism. Because the results implicate this pathway without defining precisely how it is induced, it remains possible that the same pathway could be used in a unique regulatory circuit. In either case, the lesson seems to be that the remarkable conservation of the eukaryotic cell cycle regulatory machinery is coupled with an equally remarkable flexibility in how that machinery can be deployed, depending on the particular developmental constraints of each organism. In early Drosophila embryos, a regulatory pathway that usually serves a surveillance function plays an essential cell cycle role (Price, 2000).
Deletion mutants of wee were generated by transposase-mediated imprecise excision of a nearby P{w+} transposon insertion, associated with l(2)k10413. Complementation tests have shown that all three female-sterile mutations recovered (Dwee1ES1, Dwee1ES2, and Dwee1DS1) are alleles of the same gene, and this gene corresponds to Dwee1. A detailed phenotypic analysis was undertaken of one of the mutant alleles, Dwee1ES1. Hemizygous Dwee1ES1 mutant females are viable but completely sterile and show no paternal rescue effect (hemizygous males are fertile, however). Hemizygous females lay abundant eggs of normal appearance that proceed through the early syncytial nuclear cycles without incident. During cycles 11 and 12, however, nuclei in mutant-derived embryos fail to separate at the end of mitosis and remain fused. This phenotype and the subsequent clumping and fragmentation of nuclei observed is identical to what is seen in embryos collected from grp or mei-41 mutant females. This observation suggests a possible role for Dwee1 in the same developmental checkpoint as mei-41 and grp (Price, 2000).
Two different approaches were undertaken to demonstrate that the complementation group represented by the three female-sterile mutations does in fact correspond to wee. (1) genomic DNA isolated from adults hemizygous for each of the alleles was sequenced. With respect to their maternal phenotype, Dwee1ES1 and Dwee1ES2 behave as classical amorphic alleles. Genomic DNA isolated from each of these mutants contains a DNA lesion within the kinase domain of wee that is expected to either abolish or severely disrupt the function of the gene. Dwee1ES1 contains an 8-bp deletion causing a frameshift followed by a stop codon, truncating the protein in kinase domain IV. Dwee1ES2 contains a missense mutation that changes a glutamate residue that is conserved among Wee1-like kinases to a lysine at position 308 in the protein (E308K). Dwee1DS1 behaves as a classical hypomorphic allele in that the phenotype of embryos derived from homozygous females is much less severe (many cellularize and some even develop to adulthood) than that of embryos derived from hemizygous females (which rarely cellularize and never hatch). Sequence analysis of this allele has shown that it contains a missense mutation changing a conserved phenylalanine residue to isoleucine at amino acid residue 250 within the ATP-binding site of the protein (F250I). Presumably this lesion is still compatible with low-level function of the protein. The Dwee1ES1 allele shows an antimorphic interaction with the Dwee1DS1 allele in that the phenotype of embryos derived from Dwee1DS1/Dwee1ES1 transheterozygous mothers is more severe (embryos never cellularize) than seen in Dwee1DS1/Df(2L)Dwee1WO5 hemizygotes. Conceivably, this reflects titration of positive regulatory factors by the truncated Dwee1ES1 protein, thus lowering the effective levels of Dwee1DS1 function (Price, 2000).
(2) The phenotype of mutant embryos can be partially rescued with a heat-inducible wee cDNA transgene. Maternal Dwee1ES1 hemizygous flies carrying this transgene were briefly heat-shocked to induce expression as confirmed by immunoblot analysis. Rescue was scored as development at least to the cellularization stage (cycle 14), which mutant-derived embryos otherwise never reach. By this measure, ~50% of the embryos could be rescued by maternal expression of the transgene. Cessation of heatshocks produced a decline in numbers of rescued embryos. Wide phenotypic variation was observed in the extent of phenotypic rescue, presumably reflecting variations in the amount and timing of Wee protein and mRNA deposited into individual eggs. These ranged from mosaic embryos containing both cellularized and syncytial sectors to apparently normal late embryos and first instar larvae that were nonetheless unable to complete development. A single transgene copy of a genomic DNA construct that contains wee coding sequences plus flanking DNA (and includes the adjacent dhp1-like gene) can completely rescue the maternal lethal phenotype. These two lines of evidence demonstrate that molecular lesions consistent with loss of function in wee are found in the female-sterile mutants and also show that wee expression is both necessary and sufficient to rescue the maternal lethal phenotype. It is concluded from this evidence that mutant alleles of wee have been isolated. The striking similarity between the phenotype of wee mutant-derived embryos and embryos derived from grp or mei-41 mutants provides a strong argument that maternally provided wee plays an essential role in the same developmental process as grp and mei-41 (Price, 2000).
Additional evidence in favor of this hypothesis is afforded by providing extra maternal copies of the genomic wee transgene in a mei-41D3 mutant background. Females homozygous for the mei-41D3 allele produce cellularized embyros at a very low frequency (2%). The frequency of cellularized embryos is dramatically increased by adding an extra maternal copy of a wee genomic transgene (20%). The mei-41D3 mutant embryos are further rescued by addition of two wee transgenes (50%), to the extent that some mei-41D3-derived embryos are able to develop to adulthood. In contrast, parallel experiments in a grp1 background did not produce any rescue of the mutant phenotype with either one or two extra copies of wee. The simplest interpretation that can be offered for why the results differ between grp and mei-41 mutants in these experiments is that the mei-41D3 is not a complete loss-of-function allele, and consequently mei-41D3 mutants are more sensitive to increased dosage of wee than grp1 mutants. Alternatively, grp may respond to two different signaling pathways whereas mei-41 may respond to only one of the two. Wee1 overproduction could be sufficient to rescue the common function but not the grp-specific one according to this model. Another test for functional interactions among these genes was to assess the effect of lowering the maternal dosage of mei-41+ or grp+ in a homozygous Dwee1DS1 maternal background. The incompletely penetrant syncytial arrest phenotype of homozygous Dwee1DS1-derived embryos (54% cellularized) was enhanced by subtracting a maternal copy of mei-41+ (39%). Removal of one maternal copy of grp+ produced an even greater enhancement of the mutant phenotype of Dwee1DS1 embryos (29% cellularized) (Price, 2000).
It was of interest to assess whether wee hemizygous flies derived from heterozygous parents would be capable of mounting an effective response to delays in DNA replication, since the slowing of the late syncytial cycles has been proposed to reflect activation of a DNA replication checkpoint. For this experiment, the sensitivity was assessed of Dwee1ES1 hemizygous larvae to treatment with hydroxyurea (HU; a drug that inhibits DNA replication). In fission yeast, the 'checkpoint rad' group of mutants as well as wee1 mutants are all extremely sensitive to HU. In Drosophila, mei-41 and grp mutant larvae also exhibit this response. Genetic crosses between balanced heterozygous stocks carrying either the Dwee1ES1 mutant chromosome or the Df(2L)Dwee1W05 chromosome generate both heterozygous and hemizygous viable adult progeny. Exposure to 1 or 2 mM HU eliminates the hemizygous Dwee1ES1 class of progeny, indicating that wee mutant larvae are indeed highly sensitive to HU, presumably reflecting a requirement for wee activity in a fully functional DNA replication checkpoint (Price, 2000).
Drosophila double park (dup) encodes a homolog of Cdt1 that functions in initiation of DNA replication in fission yeast and Xenopus. This study shows that mitotic checkpoint genes mei-41 and bub1 block mitosis at two distinct steps in response to incomplete DNA replication in Drosophila embryos. A study of double parked mutants demonstrate two ways in which mitosis is regulated in response to incomplete duplication of the genome: (1) entry into mitosis is delayed via mei-41 (Drosophila ATR); (2) exit from mitosis is blocked via a spindle checkpoint function, bub1. double park mutants complete the first 15 embryonic cell cycles, presumably via maternal dup products, and show defects in the 16th S phase (S16). Cells carrying dupa1 allele forgo S16 altogether but enter mitosis 16 (M16). The timing of entry into M16 is similar in dupa1 and heterozygous or wild-type (wt) controls. In contrast, mutant cells carrying another allele, dupa3, undergo a partial S16 and delay the entry into M16. Thus, initiation of S16 appears necessary for delaying M16. This delay is absent in double mutants of dupa3 and mei-41 (Drosophila ATR), indicating that a mei-41-dependent checkpoint acts to delay the entry into mitosis in response to incomplete DNA replication. dupa3 and dupa1 mutant cells that enter M16 become arrested in M16. Mitotic cyclins are stabilized and a spindle checkpoint protein, Bub1 (Basu, 1999), localizes onto chromosomes during mitotic arrest in dup mutants. These features suggest an arrest prior to metaphase-anaphase transition. dupa3 bub1 double mutant cells exit M16, indicating that a bub1-mediated checkpoint acts to block mitotic exit in dup mutants. This is the first report of (1) incomplete DNA replication affecting both the entry into and the exit from mitosis in a single cell cycle via different mechanisms and (2) the role of bub1 in regulating mitotic exit in response to incomplete DNA replication (Garner, 2001).
Yeast mutants that cannot complete DNA replication arrest before mitosis, but yeast mutants that fail to initiate DNA replication fail to arrest; thus, initiation of DNA replication appears necessary to activate a checkpoint that couples mitosis to the completion of S phase. This can account for Drosophila dup mutants that fail to undergo S16 but enter M16. It was, however, surprising to find that cells of dupa1 and dupa3 mutant embryos, previously thought to behave similarly, enter M16 at different times. dupa1 mutant cells entered M16 approximately concurrent with heterozygous controls, whereas dupa3mutant cells entered M16 after heterozygotes. While the dupa1 allele results from a stop codon at 171 (out of a total of 743 amino acids), the dupa3allele results from a stop codon at position 592. Thus, dupa3 mutants may retain partial Dup activity that allows a partial S16 and consequently activates a checkpoint to delay M16. This idea is supported by two pieces of data: (1) a partial S16 is detected in dupa3 mutants, while confirming the observation that S16 is absent in dupa1 mutants; (2) the delay of M16 seen in dupa3 mutants is found to be absent in mei-41 dupa3double mutants. mei-41 encodes a homolog of the checkpoint kinase ATR and is required to delay mitosis upon inhibition of DNA synthesis in Drosophila syncytial embryos (cycles 11-13) (Sibon, 1999). It is concluded that partial DNA synthesis in dupa3mutants delays the entry into M16 via a Mei-41-dependent checkpoint (Garner, 2001).
The effect of dup mutations on entry into mitosis is in agreement with findings in yeast and Xenopus; the effect on exit from M16, however, is contrary to previous results. Budding yeast cells that lack CDC6 skip S phase and enter mitosis (similar to dupa1mutants) but are reported to subsequently exit mitosis with wild-type kinetics to undergo a 'reductional' anaphase. In contrast, epidermal cells of both dupa3and dupa1 mutants that enter M16, with and without a prior delay, become arrested. dupa1 cells are in M16 in stage 11 and mitotic cells are still seen in stage 13; dupa3cells are in M16 in stage 13 and mitotic cells are still visible at stage 15, if not later. Because the stages are at least 2 hr apart in each case, the arrest in M16 is likely to average at least 2 hr, significantly longer than normal embryonic mitosis of about 10 min (Garner, 2001 and references therein).
The apparent difference in the behavior of yeast and Drosophila cells that harbor unreplicated chromosomes led to an effort to determine the basis for mitotic arrest in dup mutants. To this end, mitotic spindles were examined by staining for alpha- and gamma-tubulin, and chromosomes were visualized by staining for a mitotic phosphoepitope on histone H3 (PH3). While the spindles are bipolar and appear to contain functional centrosomes, i.e., they contain gamma-tubulin and nucleate aster microtubules, chromosomes fail to align normally. Most chromosomes lie within the bipolar spindle but are scattered and not compacted into a metaphase plate. Severe alignment defects are readily visible in 84% (±11%) of mitotic cells in dupa1mutants and 80% (±7%) of mitotic cells in dupa3mutants; it is probable that higher resolution analyses may reveal higher incidences of defective alignment. Because chromosome configuration in dup mutants deviates from normal configurations, other markers of mitotic progression were examined to determine at which stage in mitosis dup mutant cells arrest (Garner, 2001).
In normal mitosis, Cyclin A degradation concludes in metaphase while Cyclin B degradation concludes in early anaphase. Spindle checkpoint proteins such as Bub1 that bind kinetochores upon spindle damage localize to kinetochores during unperturbed mitosis in metazoa, indicating that the spindle checkpoint is active through earlier parts of mitosis. Drosophila Bub1 localizes on kinetochores during prometaphase and dissociates during metaphase (Basu, 1999). In dup mutants, both cyclins persist during mitotic arrest, and Bub1 is present on discrete sites on chromosomes, presumably at kinetochores. These data suggest that dup mutant cells arrest prior to metaphase-anaphase transition (Garner, 2001).
The persistence of Bub1 on chromosomes and stabilization of Cyclin B occurs when the spindle checkpoint is active; therefore, mitotic arrest in dup mutants may be mediated by the spindle checkpoint. To test this directly, double mutants of dupa3and bub1 were examined. dupa3bub1 double mutants have fewer mitotic cells when compared to dupa3 single mutant embryos of similar stage. Two types of additional evidence indicate that this difference is due to dupa3bub1 double mutants exiting M16 (rather than reverting to previous interphase). (1) Approximately 10 times more cells are seen in the act of exiting mitosis (i.e., in telophase) in the double mutants. Most telophase cells show chromosome bridges, consistent with the failure to complete DNA replication in these mutants. (2) Nuclear density is higher in dupa3bub1 double mutants than in dupa3 single mutants, and it approaches that of heterozygotes or wt controls that complete M16. Collectively, these data indicate that a significant number of dupa3bub1 mutant cells exited M16 and suggest that bub1 is required for mitotic arrest in dup mutants (Garner, 2001).
During mitotic arrest by the spindle checkpoint, Cyclin A is degraded while Cyclin B remains stable. Therefore, persistence of Cyclin A during mitotic arrest in dup mutants suggests that additional control(s), in addition to the spindle checkpoint, operate to stabilize Cyclin A. DNA damage leads to stabilization of Cyclin A in Drosophila. Possibly, the presence of incompletely replicated DNA during mitosis also leads to stabilization of Cyclin A. bub1-mediated controls, however, appear more consequential because dupa3bub1 double mutants exited mitosis. This would be consistent with the finding that Cyclin A at endogenous levels cannot block mitotic exit in Drosophila (Garner, 2001).
CDC6-deficient cells are reported to exit mitosis with normal kinetics. An examination of these cells, however, reveals a mitotic arrest that requires MAD2. Thus, incomplete DNA replication in both yeast and Drosophila results in mitotic arrest mediated by members of the spindle checkpoint. Why might this be? A complete or partial absence of sister chromosomes would lead to a complete or partial absence of sister chromatid cohesion. The failure to duplicate centromeres, likely in dupa3mutants and certainly in dupa1mutants, would preclude the formation of kinetochore pairs. Either deficiency would preclude stable bipolar attachment of chromosomes to the spindle and thereby activate the spindle checkpoint (Garner, 2001).
In summary, dup mutants demonstrate two ways in which mitosis is regulated in response to incomplete duplication of the genome: (1) entry into mitosis is delayed via mei-41, Drosophila ATR; (2) exit from mitosis is blocked via a spindle checkpoint function, bub1. In Drosophila syncytial cycles, nuclei delay the entry into mitosis upon inhibition of DNA synthesis, but exit from mitosis is not blocked. Instead, chromosome separation fails during the exit from mitosis, resulting in polyploid nuclei that are subsequently eliminated. In other systems, either the entry into mitosis (in fission yeast and vertebrate cells) or the exit from mitosis (in budding yeast) is blocked in response to incomplete DNA synthesis. Therefore, this is the first report of mitosis in a single cell cycle being regulated at two points via two different mechanisms in response to incomplete DNA replication. Identification of these responses in Drosophila, a genetically tractable organism with superb cytology, should enable testing of candidate checkpoint genes and searching for new genes that function at each regulatory point (Garner, 2001).
The COP9 signalosome (CSN) is linked to signaling pathways and ubiquitin-dependent protein degradation in yeast, plant and mammalian cells, but its roles in Drosophila development are just beginning to be understood. During oogenesis, one subunit of the CSN [COP9 complex homolog subunit 5 (CSN5/JAB1)], is required for meiotic progression and for establishment of both the AP and DV axes of the Drosophila oocyte. CSN5 mutations block the accumulation of the Egfr ligand Gurken in the oocyte, interfering with axis formation. CSN5 mutations also cause the modification of Vasa, which is known to be required for Gurken translation. This CSN5 phenotype (defective axis formation, reduced Gurken accumulation and modification of Vasa) is very similar to the phenotype of the spindle-class genes that are required for the repair of meiotic recombination-induced DNA double-strand breaks. When these breaks are not repaired, a DNA damage checkpoint mediated by mei-41 is activated (Ghabrial, 1999). Accordingly, the CSN5 phenotype is suppressed by mutations in mei-41 or by mutations in mei-W68, which is required for double-strand break formation. These results suggest that, like the spindle-class genes, CSN5 regulates axis formation by checkpoint-dependent, translational control of Gurken. They also reveal a link between DNA repair, axis formation and the COP9 signalosome, a protein complex that acts in multiple signaling pathways by regulating protein stability (Doronkin, 2002).
Because of the similarity between the CSN5 and spindle-class phenotypes, a connection between CSN5 and the meiotic checkpoint mediated by mei-41 was tested. The viable hypomorphic combination CSN5ex21/CSN5L4032 causes a reduction in Grk protein level, especially during the early stages of oogenesis. Five to fifteen percent of eggs laid by these transheterozygotes had fused dorsal appendages, indicating a partial reduction of Grk. When CSN5ex21/CSN5L4032 flies were also homozygous-mutant for mei-41, however, the normal Grk protein level was restored, and the eggshell phenotype was rescued (Doronkin, 2002).
Interestingly, checkpoint activation leads to modification of the Vasa protein, as shown by a slightly reduced mobility during SDS polyacrylamide gel electrophoresis. This result is relevant to the spindle-class and CSN5 phenotypes because Vasa regulates translation of Gurken and, as a consequence, axial patterning. This Vasa modification is checkpoint dependent since it is present in spn-B mutants but absent in mei-41 spn-B double mutants (Doronkin, 2002 and references therein).
A similar reduced mobility of Vasa protein is detected in CSN5 mutants. For viable CSN5 mutants there were two Vasa bands: one corresponding to Vasa from wild-type ovaries and a second with lower mobility. In stronger mutant combinations, most of the Vasa protein was modified, while in weaker combinations most Vasa had normal mobility. The shift in Vasa mobility was suppressed by mei-41 mutations. Interestingly, removal of one dose of mei-41 completely restores normal Vasa mobility for a weak CSN5 combination. For stronger CSN5 mutants, full restoration of Vasa mobility requires removal of both mei-41 genes (Doronkin, 2002).
The gene mei-W68 is required for the initiation of meiotic recombination in Drosophila ovaries and is likely to induce DNA double-strand breaks (DSBs) as recombination begins. Mutations in mei-W68 rescue spindle-class defects, including Grk protein accumulation, eggshell morphology and Vasa modification. These results suggested that since DSBs are not formed in the absence of mei-W68, DNA repair by the spindle-class genes is not required. A similar interaction is seen between mei-W68 and CSN5. Hetrerozygosity for mei-W68 is sufficient to suppress the phenotypes of both strong and weak CSN5 allelic combinations (Doronkin, 2002 and references therein).
These data demonstrate that absence of CSN5 function during meiosis activates a DNA-damage checkpoint that is mediated by Mei-41. Because the reduction in DSBs in mei-W68 heterozygotes removes the requirement for CSN5, it is likely that CSN5 promotes DNA repair, as do the spindle-class genes (Doronkin, 2002).
The mei-41RT1 allele contains a single nonautonomous P-element transposon inserted in the mei-41 coding region. Following mobilization of this transposon, 454 chromosomes were recovered and tested for female sterility when heterozygous with the mei-41D3 allele. A total of 392 recovered chromosomes caused reduced female fertility when heterozygous with the mei-41D3 allele, similar to mei-41D3/mei-41RT1 control females. Thirty-three chromosomes were female fertile when heterozygous with the mei-41D3 allele and thus may represent revertants. Twenty-nine exhibited female sterility when heterozygous with mei-41D3. One of these 29, 78B, carries a homozygous lethal mutation and was rescued by a chromosomal duplication, Dp(1;4)r+f+, that includes mei-41. However, 78B fails to complement lethal mutations at l(1)14Ce locus (alleles 4d25 and 4a27) and therefore is likely to carry a deficiency (Laurençon, 2003).
Genomic DNA from the these 29 mutants was amplified by PCR and analyzed. Three mutants, 29D, 99B, and deficiency-bearing 78B, were found to lack the P element. Further sequence analysis showed that the 29D chromosome is missing 975 bp of mei-41 coding sequence. Moreover, transposon excision was accompanied by the insertion of an extra A, creating a frameshift in the remaining coding sequence. Consequently, the 40th codon is expected to encode a 'stop', leaving a truncated protein of 39 aa; the native mei-41 gene encodes 2347 aa. The 99B line presents an 8-bp direct duplication with most of the P-element sequence deleted, leaving behind 13 bp of 5' inverted repeat (IR) and 17 bp of the 3' IR that are separated by nucleotides, CA. This rearrangement creates a stop codon downstream of the insertion site and is predicted to encode a truncated protein of 340 aa. 29D and 99B are thus null mutants; these new alleles are referred to as mei-4129D and mei-4199B (Laurençon, 2003).
Despite the severe nature of lesions, homozygous females and hemizygous males bearing mei-4129D or mei-4199B alleles are fully viable. Thus null alleles of mei-41 do not appear to act as zygotic lethal mutations. Note that the recovery of the 78B deficiency indicates that lethal alleles of mei-41 would have been recovered had they been induced. In mammals, ATR is an essential gene while ATM is not (Barlow, 1996; Xu, 1996; Brown, 2000). The Drosophila genome project identified a second ATM/ATR homolog in the Drosophila genome (CG6535), which is more closely related to ATM; Mei-41 is more closely related to ATR. It is therefore interesting that mei-41 appears to be nonessential, in contrst to the essential role of ATR (Laurençon, 2003).
Prior to further analysis, it was first confirmed that newly generated null alleles exhibit phenotypes expected of strong mei-41 alleles. In assays for sensitivity to a genotoxin (MMS), doses as low as 0.01% killed 100% of homozygotes for the mei-4129D and mei-4199B alleles. Moreover, mei-4129D homozygous females are semisterile (97.3% of embryos fail to hatch at 25°C. and 92% at 20°C.) and display meiotic defects such as reduction of recombination (13% of normal between net and ho markers) and increased chromosome losses and nondisjunction (10% X chromosome loss; control shows <1%). Likewise, the ability to delay the entry into mitosis upon DNA damage is disrupted in homozygous mei-4129D mutant larvae. These phenotypes are identical to those caused by the strongest mei-41 alleles isolated to date, namely D1, D3, and 195 (Laurençon, 2003).
A semidominant effect on MMS sensitivity has been repored for at least one strong allele of mei-41. mei-4129D and mei-4199B also exhibit a semidominant effect on larval MMS sensitivity and the ability to block mitosis after DNA damage in larval discs. However, female sterility does not appear to be dose dependent since one copy of the mei-41 genomic DNA can rescue embryonic lethality to wild-type levels (Laurençon, 2003).
mei-4129D mutants were used to determine the role of mei-41 in a recently defined metaphase checkpoint in embryos (Su, 2001). In embryonic cell cycles, DNA damage due to ionizing radiation or MMS causes a delay in the entry into mitosis. After this delay, cells recover but subsequently are delayed in metaphase/anaphase transition (Su, 2001). This is seen in live measurement of mitotic timing, where duration of metaphase increases about threefold, as well as in quantification of mitotic phases in fixed embryos; the ratio of metaphase to {anaphase + telophase} increases by about threefold in irradiated embryos (Su, 2001). Metaphase/anaphase delay appears to be due to stabilization of a mitotic cyclin, but the role of checkpoint genes in this response has not been addressed (Laurençon, 2003).
The number of mitotic cells in the dorsal ectoderm is reduced at 20 min after irradiation in wild type and in homozygous mei-4129D embryos from heterozygous mothers, but not in homozygous mei-41D12 embryos from homozygous mei-41D12 mothers. There are several possible explanations for these observations. Homozygous mei-4129D embryos would have inherited wild-type mei-41 gene products from their heterozygous mothers, and the maternal product may persist long enough to enforce the mitotic entry checkpoint; in contrast, D12 embryos from homozygous D12 mothers would not have inherited any wild-type mei-41 gene products. It is also possible that mei-41 is dispensable for the regulation of mitotic entry after X-ray damage in embryos or that it plays a redundant role with a second ATM/ATR homolog that exists in Drosophila (Laurençon, 2003).
More important for this work, at longer times (40 min) after irradiation when cells have recovered and enter mitosis, the ratio of metaphase to {anaphase + telophase} increases in wild type; this increase is severely diminished in mei-4129D and mei-41D12 embryos and partially diminished in mei-41D13 embryos. Thus, all three mei-41 alleles tested are defective in the metaphase/anaphase checkpoint, implicating this gene in the regulation of mitotic progression in response to X-ray damage (Laurençon, 2003).
mei-4129D embryos have an intact mitotic entry checkpoint but not an intact metaphase/anaphase checkpoint. This could be either because the latter is more sensitive to the level of maternal mei-41 gene products that persists or because maternal mei-41 products are able to substitute for zygotic mei-41 products in the first checkpoint but not in the second. Thus, the data implicate mei-41 in the regulation of metaphase/anaphase transition after DNA damage but do not conclusively address the role of mei-41 in the embryonic mitotic entry checkpoint. Nonetheless, metaphase/anaphase regulation is defective in mei-41 mutants that are either able (29D) or unable (D12) to regulate the entry into mitosis. This result excludes the possibility that metaphase/anaphase defects are due to variations in the time of entry into mitosis between wild type and mutants (Laurençon, 2003).
ATR homologs act as signal transducers in DNA replication and damage checkpoint pathways. The presence of DNA defects activates ATR homologs, which are kinases that in turn carry out their function by phosphorylation of downstream substrates. Drosophila ATM/ATR, Mei-41, when mutated, leads to a number of phenotypes as described above, ranging from female sterility to MMS sensitivity. If the same checkpoint pathway and components are at work in processes whose failure generates these phenotypes, it might be expected that an allele that affects one phenotype will affect others to a similar extent. However, if the function of mei-41 is being executed via different partners/substrates in different processes, it might be expected that an allele that affects one phenotype may not necessarily affect another (Laurençon, 2003).
To address these possibilities, the phenotypes of 7 previously isolated alleles were compared to one another and to the null allele. Five (D9, D12, D13, D14, and D15) were chosen out of 33 tested alleles because they behave as wild type in two standard assays for meiotic functions, namely X chromosome nondisjunction frequency and meiotic recombination levels. Thus, the contribution of meiotic defects to female sterility can be ruled out. The sixth, D5, was selected because it affects partially all mei-41 phenotypes described in this study; mutant females are meioticaly impaired, showing 5% of X nondisjunction. Finally, D3 was selected because it behaved as a null and thus a mutation abolishing all mei-41 functions is likely to be present. Additionally, because all 7 alleles were generated during a single EMS mutagenesis, it was reasoned that they will share a common background and therefore sequence analysis would reveal mutations affecting mei-41 functions (Laurençon, 2003).
A comparison of mei-41 alleles leads to two conclusions. (1) Not all phenotypes are affected equally by each allele. For instance, D12 and D13 alleles present wild-type levels of female fertility but defective metaphase/anaphase checkpoints in the embryo and defective mitotic checkpoints in larval discs. Conversely, the larval mitotic checkpoint is as robust in D15 as in wild type, even though D15 mutants are MMS sensitive and partially female sterile. (2) An allele that is more defective than another with regard to one phenotype is not necessarily so with regard to another phenotype. For instance, the D14 allele is more severe than D9 in female fertility but is less severe than D9 in the mitotic checkpoint in larval discs. Likewise, D12 shows less severe female fertility and MMS sensitivity than does D15, but is more severe than D15 in larval mitotic checkpoint regulation. These observations raise the possibility that some aspects of the mei-41 phenotype may reflect the specific mutational ablation of domains critical for a specific function. To address this, the sequence of the mutant alleles was determined (Laurençon, 2003).
Sequence analysis reveals several differences between mutant alleles and the published mei-41 sequence. Seventy silent mutations were found, 12 of which are shared by all seven mutant alleles, and 10 mutations were found leading to amino-acid changes, all of which are shared by two or more alleles. These deviations could be due to variability originally present in the mutagenized population (as indicated by common mutations within each of two different groups: D3 and D14 in one group and D5, D9, D12, D13, and D15 in the other and/or accumulation of mutations in mei-41 mutants. In addition to these common mutations, a single unique mutation was found for each allele and may account for the phenotype. For mei-41D3, a T --> A change at nucleotide 3768 is predicted to convert a nonpolar A to a nonpolar V. Additional mutations are postulated in the noncoding region for mei-41D3 because very little protein is detected in these mutants (Sibon, 1999). The mei-41D5 mutation changes proline2159 in the kinase domain to a leucine. This proline is conserved among all members of the PI3K-L family except for UVSB (which has a T) and Ce-atl-1 (which has an A). mei-41D5 shows a partial defect in all assayed phenotypes, suggesting that kinase activity of Mei-41 is important for all its functions. Likewise, mutations in the kinase domain (Bentley, 1996; Cliby, 1998; Martinho, 1998) affect all functions known for RAD3 and ATR (Laurençon, 2003).
Interestingly, unique mutations in the other five alleles analyzed in this study fall outside of the kinase domain. Among PI3K-l family members, there is little sequence similarity outside of the kinase domain. The exception is a computationally defined region of ~500 amino acids, called the FAT domain that is shared with the TRRAP family of proteins (Bosotti, 2000). This domain is a part of the rad3 domain, which is conserved only in a subgroup that includes Mec1p, Rad3, ATR, and Mei-41, but not ATM. The mei-41D12 mutation affects a conserved histidine within the rad3/FAT domain. mei-41D13 is a mutation in the N terminus where an algorithm designed to detect DNA-binding domains detects a putative helix-turn-helix. mei-41D9, mei-41D14, and mei-41D15 affect a predicted alpha-helix in the N terminus. The mei-41D14 and mei-41D15 mutations change, respectively, a leucine to a phenylalanine and a threonine to a proline and either is likely to disorganize the putative alpha-helix. mei-41D9 mutants show a more severe defect in larval mitotic checkpoint than do mei-41D14 and mei-41D15 mutants; this suggests that the predicted change of a reactive serine647 to a hydrophobic phenylalanine in mei-41D9 may not only destabilize the putative helix but also specifically disrupt interaction(s) in which this helix participates. In sum, the sequence analysis of partial loss-of-function alleles suggests the importance of N-terminal sequences and identifies mutations that presumably affect one phenotype more severely than another (Laurençon, 2003).
Cells of metazoan organisms respond to DNA damage by arresting their cell cycle to repair DNA, or they undergo apoptosis. Two protein kinases, ataxia-telangiectasia mutated (ATM) and ATM and Rad-3 related (ATR), are sensors for DNA damage. In humans, ATM is mutated in patients with ataxia-telangiectasia (A-T), resulting in hypersensitivity to ionizing radiation (IR) and increased cancer susceptibility. Cells from A-T patients exhibit chromosome aberrations and excessive spontaneous apoptosis. Drosophila has been used as a model system to study ATM function. Previous studies suggest that mei-41 corresponds to ATM in Drosophila; however, it appears that mei-41 is probably the ATR ortholog. Unlike mei-41 mutants, flies deficient for the true ATM ortholog [telomere fusion (common alternative name: ATM)] die as pupae or eclose with eye and wing abnormalities. Developing larval discs exhibit substantially increased spontaneous chromosomal telomere fusions and p53-dependent apoptosis. These developmental phenotypes are unique to Drosophila ATM, and both Drosophila ATM and mei-41 have temporally distinct roles in G2 arrest after IR. Thus, ATM and ATR orthologs are required for different functions in Drosophila; the developmental defects resulting from absence of Drosophila ATM suggest an important role in mediating a protective checkpoint against DNA damage arising during normal cell proliferation and differentiation (Song, 2004).
dATM, mei-41, grp, and dChk2 are close orthologs of mammalian DNA damage checkpoint genes that are all highly expressed in Drosophila females relative to males. The encoded checkpoint protein kinases presumably respond to DNA double-strand breaks generated during meiotic recombination. Notably, meiotic recombination in Drosophila occurs only in females. Moreover, mammalian ATM and ATR localize to synapsed and unsynapsed regions of meiotic chromosomes, respectively, suggesting a role for these proteins during meiotic recombination. In Drosophila, most mei-41 alleles are recessive female sterile, and females homozygous for the more fertile hypomorphic mei-41 alleles exhibit reduced meiotic exchange. The Δ356 dATM mutant females do not lay eggs, whereas Δ11 females lay a very small number of eggs that do not hatch, indicating that dATM is required for female fertility. In mouse models, ATR is an essential gene, whereas ATM is dispensable for normal development and viability, although homozygous mutant mice exhibit immune defects, growth retardation, and sterility. In Drosophila, the mei-41(ATR) mutant was found to be maternal lethal and nullizygous dATM mutants were found to be lethal. Thus, unlike its mammalian counterpart, Drosophila ATM plays a critical developmental role (Song, 2004).
To examine the temporal roles of ATM and mei-41 in Drosophila, mitosis (as an indicator of cell cycle arrest) was examined at various time points after irradiation. Wild-type wing discs had very few mitotic cells 25 min after irradiation, and no mitotic cells were detected at 1 hr postirradiation. The wing disc cells remained arrested and then reentered the cycle approximately 6 hr later. Interestingly, dATM mutant wing discs had significantly more mitotic cells 25 min postirradiation as compared to wild-type discs. At later time points, G2 arrest occurred normally in dATM mutant wing discs. In mei-41 mutant wing discs, cells continued to cycle throughout the time points tested. These results suggest that dATM is involved in the early phase of G2 arrest and mei-41 has a major role in late response. The observed temporal differences in the IR-mediated checkpoint are consistent with those found in mammals (Song, 2004).
Potential functional redundancy between dATM and mei-41 was examined by generating double mutants. Flies harboring mutations in both genes exhibit the same excessive level of spontaneous apoptosis seen in the dATM mutant larval tissues and no apparent increase in apoptosis following IR; however, most of the third-instar larvae exhibited black 'growths', characteristic of so-called melanotic tumors. The underlying basis for these growths is not known, but it suggests that there is some developmental context in which these two genes function redundantly. The mechanisms underlying the distinct and overlapping functions of dATM and mei-41/ATR are likely to involve the upstream activation signals and downstream effector substrates for these closely related kinases; as such, the Drosophila model provides a genetically tractable system that should prove useful in dissecting their function in an in vivo context (Song, 2004).
In conclusion, the function of Drosophila ATM was examined and its role was compared with that of mei-41/ATR during normal development and DNA damage checkpoint responses. Both dATM and mei-41 are highly expressed in female adults and are required for female fertility. Unlike mei-41, flies deficient for dATM exhibit a substantial increase in spontaneous p53-dependent apoptosis and telomere fusions in developing tissues, leading to lethality or to viable flies with malformed adult tissues. It is presumed that dATM deficiency leads to the accumulation of DNA damage during normal cellular replication and differentiation and that this culminates in p53 activation. Although some ATM functions in mammals are mediated by the Chk2 kinase, the essential developmental role of dATM is apparently not mediated by dChk2, which is a nonessential gene, indicating that other ATM substrates are required. In addition to their distinct developmental requirements, dATM and mei-41/ATR perform temporally distinct functions in the DNA damage response to ionizing radiation. Together with the characterization of ATM and ATR functions in mammalian systems, these studies of the Drosophila orthologs point to evolutionarily conserved pathways involving two closely related proteins that together regulate genomic integrity during normal development and in response to genotoxic stress (Song, 2004).
Components of the DNA damage checkpoint are essential for surviving exposure to DNA damaging agents. Checkpoint activation leads to cell cycle arrest, DNA repair, and apoptosis in eukaryotes. Cell cycle regulation and DNA repair appear essential for unicellular systems to survive DNA damage. The relative importance of these responses and apoptosis for surviving DNA damage in multicellular organisms remains unclear. After exposure to ionizing radiation, wild-type Drosophila larvae regulate the cell cycle and repair DNA; grp (DmChk1) mutants cannot regulate the cell cycle but repair DNA; okra (DmRAD54) mutants regulate the cell cycle but are deficient in repair of double strand breaks (DSB); mei-41 (DmATR) mutants cannot regulate the cell cycle and are deficient in DSB repair. All undergo radiation-induced apoptosis. p53 mutants regulate the cell cycle but fail to undergo apoptosis. Of these, mutants deficient in DNA repair, mei-41 and okra, show progressive degeneration of imaginal discs and die as pupae, while other genotypes survive to adulthood after irradiation. Survival is accompanied by compensatory growth of imaginal discs via increased nutritional uptake and cell proliferation, presumably to replace dead cells. It is concluded that DNA repair is essential for surviving radiation as expected; surprisingly, cell cycle regulation and p53-dependent cell death are not. It is proposed that processes resembling regeneration of discs act to maintain tissues and ultimately determine survival after irradiation, thus distinguishing requirements between muticellular and unicellular eukaryotes (Jaklevic, 2004).
In eukaryotes, DNA damage checkpoints monitor the state of genomic DNA and delay the progress through the cell cycle as needed. Central components of this checkpoint in mammals include four kinases: ATM, ATR, Chk1, and Chk2. Homologs of these exist in other eukaryotes and assume similar roles where examined. Human patients with ATM mutations, as well as their cells, show a dramatic sensitivity to killing by ionizing radiation. The importance of checkpoints in cellular survival to DNA damaging agents is presumed to be due to the role of checkpoints in cell cycle regulation. This is because mutants in the budding yeast gene rad9, the first checkpoint gene to be characterized, fail to arrest the cell cycle following damage and show increased radiation sensitivity; the latter phenotype is rescued by experimental induction of cell cycle delay. Consequently, cell cycle delay is thought to allow time for DNA repair and thereby ensure survival (Jaklevic, 2004 and references therein).
Components of the DNA damage checkpoint are found to activate DNA repair and to promote programmed cell death, which would cull cells with damaged DNA. For example, phosphorylation of NBS (a component of the Mre11 repair complex) by human ATM is of functional importance, while ATM knockout mice show a reduction in radiation-induced cell death in the CNS. Therefore, the essential role of checkpoints in conferring survival to genotoxins may be due to DNA repair and cell death responses in addition to or instead of cell cycle regulation. Furthermore, what is important for survival at the cellular level may not be so in a multicellular context. For instance, the failure to arrest the cell cycle by checkpoints may be detrimental to individual cells, but removal of these by cell death and replacement via organ homeostasis may make cell cycle regulation inconsequential for survival of multicellular organs (Jaklevic, 2004).
To address how DNA damage checkpoints operate in the context of multicellular organisms in vivo, the effect of ionizing radiation on Drosophila melanogaster is being studied. In Drosophila, mei-41 (ATR homolog) and grp (Chk1 homolog) are required to delay the entry into mitosis in larval imaginal discs after irradiation and to delay the entry into mitosis after incomplete DNA replication in the embryo. Thus, mei-41 and grp play similar roles to their homologs in other systems. Moreover, mei-41 mutants are deficient in DNA repair. The role of mei-41 and grp in radiation-induced cell death has not been tested, but mei-41 is dispensable for cell death after enzymatic induction of DNA double-strand breaks (Jaklevic, 2004 and references therein).
Mutants in mei-41, grp, p53, and okra, a homolog of budding yeast RAD54 that functions in repair of DNA double-strand breaks (DSB) have been used to address the relative importance of cell cycle regulation, cell death, and DNA repair to the ability of a multicellular organism to survive ionizing radiation. The three responses are affected to different degrees in these mutants: wild-type larvae regulate S and M phases and repair DNA; grp mutants are unable to regulate the cell cycle but are able to repair DNA; okra mutants are able to regulate the cell cycle but are deficient in DNA repair; and mei-41 mutants are unable to regulate the cell cycle and are also deficient in DNA repair. All genotypes with the exception of p53 mutants are proficient in radiation-induced cell death, suggesting that mei-41 and grp do not contribute to this response. Under these conditions, it is found that while mei-41 and okra mutants are highly sensitive to killing by ionizing radiation, p53 mutants show reduced but significant survival and grp mutants resemble wild-type. These results suggest that cell death is neither sufficient nor absolutely necessary, DNA repair is essential, and optimal cell cycle regulation is dispensable for surviving ionizing radiation in Drosophila larvae (Jaklevic, 2004).
The effects of DNA damage by ionizing radiation on the maintenance and survival of Drosophila larvae was studied. Despite an extensive loss of cells to radiation-induced cell death, organ size and morphology are maintained remarkably well, and larvae survive to produce viable adults. Surprisingly, optimal cell cycle regulation by checkpoints is neither necessary (as in grp mutants) nor sufficient (as in okra mutants) to ensure organ homeostasis and organismal survival. p53-dependent cell death is also largely dispensable in this regard. Instead, DNA repair appears to be of paramount importance as might be expected (Jaklevic, 2004).
In mitotically proliferating cells of Drosophila larval imaginal discs and brains, the first responses to sublethal doses of irradiation (1000R-4000R) are delays in cell cycle progression at 1-2 hr after irradiation, followed by the induction of cell death at 4 hr after irradiation. DNA synthesis resumes at 5 hr after irradiation, while mitotic index resumes at 6 hr after. These relatively early responses are followed by an increase in proliferation that is detectable about a day after irradiation. Presumably, abundant cell death removes damaged cells, but sustained proliferation compensates to maintain proper organ size and morphology. Continued cell proliferation, it is proposed, delays the onset of the next major developmental transition, pupariation. The extent of delay correlates with radiation dose, presumably because more cells are lost at higher doses, requiring more compensatory proliferation (Jaklevic, 2004).
Another response monitored was DNA repair, a substantial portion of which must occur within 3 hr after 220R of irradiation because a significant difference is seen in the incidence of chromosome breakage between wild-type and repair-deficient mutants by this time. However, cytologically visible chromosome breaks likely represent only a fraction of total DNA damage; for this reason, it is not certain if DNA repair is complete within this time frame (Jaklevic, 2004).
Having determined the sequence of responses to irradiation in wild-type larvae, deviations from it in various mutants was documented. mei-41 and grp mutants are unable to dampen DNA synthesis after irradiation. Previous work has shown that both mutants are unable to inhibit mitosis after irradiation, although grp mutants appear to retain a partial activity in this regard. Thus, Drosophila ATR and Chk1 are needed for optimal regulation of both S and M phases after exposure to ionizing radiation. However, induction of cell death does not require mei-41 or grp. The most striking result report in this study is that grp mutants that are defective in regulation of both S and M phases are not sensitive to killing by 2000R of X-rays, doses that readily killed mei-41 and okra mutants. This finding strongly suggests that cell cycle regulation by checkpoints is not absolutely necessary for surviving irradiation under these conditions (Jaklevic, 2004).
In determining what is necessary, the phenotype of okra mutants that can regulate both S and M phases and promote cell death is particularly informative because they are radiation sensitive. Thus, DNA repair is essential, suggesting that it is this defect in mei-41 mutants that renders them radiation sensitive. It is speculated that irradiated mei-41 and okra larvae may attempt to increase proliferation, but the continual presence of unrepaired DNA likely channels these cells to death. This would lead to an eventual decline in cell number, which would undermine maintenance of cellular differentiation that is the basis of the eye disc's morphogenetic furrow (MF). Signals from cells in the MF are thought to be important for the generation of the second mitotic wave. Loss of the MF could then explain the absence of the expected pattern of mitoses in mei-41 and okra discs (Jaklevic, 2004).
Traditionally, checkpoints refer to the regulation of the cell cycle. Recent views propose the inclusion of the other responses among checkpoint responses, such as the preservation of DNA replication intermediates, transcriptional activation, and DNA repair. The data suggest that other responses may be more important in ensuring survival of multicellular organs and organisms. Interestingly, results from budding yeast also question the idea that cell cycle regulation by checkpoints is essential for surviving genotoxins even at the cellular level. For example, yeast Chk1 mutants show profoundly defective regulation of mitosis after irradiation and yet are only mildly radiation sensitive. Another recent study indicates that stabilization of replication forks is crucial for surviving the alkylating agent MMS whereas the ability to inhibit mitosis is less important (Jaklevic, 2004).
It is emphasized that survival in this study refers to that of organs and organisms. At the cellular level, cell cycle regulation by checkpoints may well be crucial to allow time for DNA repair and for survival. In grp mutants that are defective for cell cycle checkpoints but are proficient for DNA repair, cells that progressed through S and M phases with damaged DNA may have been subject to cell death. Indeed, incidence of cell death appears higher in grp (and mei-41) mutants than in wild-type. Loss of these cells, however, is clearly of little consequence to survival of imaginal discs and larvae. This could be because grp mutants are able to repair DNA in cells that are not in S and M phases, i.e., those in G1 or G2. These cells may then proliferate to compensate for lost cells. Numerous studies on tissue regeneration demonstrate the power of Drosophila larvae to restore not only cell number but also proper differentiation. In such a system, the failure of cell cycle checkpoints after irradiation may be of little consequence as long as damaged cells are replaced. It is speculated that these findings may be particularly applicable to multicellular systems with similar regenerative powers such as the human liver (Jaklevic, 2004).
In higher eukaryotes, the ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) checkpoint kinases play distinct, but partially overlapping, roles in DNA damage response. Yet their interrelated function has not been defined for telomere maintenance. The two proteins control partially redundant pathways for telomere protection in Drosophila: the loss of ATM (encoded by telomere fusion) leads to the fusion of some telomeres, whereas the loss of both ATM and ATR (encoded by mei-41) renders all telomeres susceptible to fusion. The ATM-controlled pathway includes the Mre11 and Nijmegen breakage syndrome complex but not the Chk2 kinase, whereas the ATR-regulated pathway includes its partner ATR-interacting protein but not the Chk1 kinase. This finding suggests that ATM and ATR regulate different molecular events at the telomeres compared with the sites of DNA damage. This compensatory relationship between ATM and ATR is remarkably similar to that observed in yeast despite the fact that the biochemistry of telomere elongation is completely different in the two model systems. Evidence is provided suggesting that both the loading of telomere capping proteins and normal telomeric silencing require ATM and ATR in Drosophila and it is proposed that ATM and ATR protect telomere integrity by safeguarding chromatin architecture that favors the loading of telomere-elongating, capping, and silencing proteins (Bi, 2005).
This study defines two partially redundant pathways, regulated by ATM and ATR, respectively, that ensure complete protection of Drosophila telomeres. It is further suggested that two other proteins, Meiotic recombination 11 (Mre11) and Nbs) belong to the same ATM-regulated pathway, whereas ATR-Interacting Protein (ATRIP or Mutagen-sensitive 304) participates in the ATR-controlled pathway. This conclusion would be consistent with the pathway components defined in yeasts. Based on the facts that Drosophila tefu mutants have widespread telomere fusions that eventually lead to lethality, whereas mei-41 mutants are viable with no apparent telomeric defects, it is proposed that ATM is more important in regulating telomere protection, with ATR playing a backup role. In mei-41 mutants, ATM may fully compensate ATR's absence on telomeres to ensure complete capping throughout the cell cycle. In contrast, ATR in tefu mutants may partially compensate for the loss of ATM because it is a less efficient regulator of capping as suggested earlier. This partial redundancy leads to the fusion of some telomeres (Bi, 2005).
ATM, ATR, and their cofactors are known to control multiple checkpoints in response to DNA damage and abnormal telomeres. Perhaps cells with uncapped telomeres are allowed to continue cycling because of defective checkpoints and that leads to the fusion of these telomeres. This possibility is considered unlikely based on several observations. (1) In cav1 mutant (see caravaggio), telomere fusion occurs at a high rate even in the presence of a full complement of checkpoint genes. (2) No exacerbated telomere dysfunction is found in either the cav1 tefu (atm) or the cav1 mei-41 (atr) double mutant. (3) Mutations in Drosophila chk1 or chk2 did not affect existing telomere defects in either tefu or mre11 mutant, suggesting that checkpoints jointly controlled by these effectors and the respective upstream kinases do not normally respond to dysfunctional telomeres. Therefore, the contribution to the telomere dysfunction from checkpoint defects is likely small in the cases studied (Bi, 2005).
Cytological analyses suggest that one of the functions of ATM and ATR at Drosophila telomeres is to facilitate the loading of telomere capping and silencing proteins, such as HOAP (Caravaggio) and HP1, and they do so in a partially redundant fashion. In the case of HOAP's binding to mitotic telomeres, either ATM or ATR is sufficient for its normal loading. When both kinases are absent, HOAP can no longer be detected at the telomeres. It was interesting that there were more telomere fusions in either tefu mei-41D9 or tefu mus304D1/D3 than in either mre11 or nbs1 cells, yet normal HOAP signals were detected on fusion-free telomeres for only the first two genotypes. It is known that the presence of HOAP at chromosome ends is not sufficient to prevent fusion, which suggests that the loss of other capping proteins could also lead to fusion. That may be the case in tefu mei-41D9 and tefu mus304D1/D3 cells. It is also known that the absence of HOAP at any particular telomere does not necessarily lead to fusion, suggesting other capping proteins can sometimes compensate for HOAP's function. That idea, in contrast, may apply to the situation in cells deficient for the Mre11 complex (Bi, 2005).
The mechanism by which ATM and ATR maintain telomere integrity remains largely unclear. Because the conserved kinase domain of both ATM and ATR is essential for normal telomere function in S. cerevisiae, ATM and ATR likely exert their function by protein phosphorylation. Neither Chk1 nor Chk2 was involved in telomere protection in Drosophila, a situation similar to S. pombe, which suggests that ATM and ATR modify a different set of proteins at telomeres. ATM and ATR may regulate HOAP's capping activity by directly phosphorylating HOAP. However, no evidence was recovered that HOAP is phosphorylated in WT cells. Therefore, ATM and ATR may indirectly regulate HOAP's ability to bind telomeres, perhaps by modulating telomeric structure. The results support the hypothesis that ATM and ATR have a conserved function at the telomeres that is independent of telomerase. Because Drosophila telomeres are not elongated by a telomerase, the fly may be an excellent system for studying the roles of ATM and ATR in telomere protection, uncoupled from their roles in telomere elongation (Bi, 2005).
Two protein kinases ATM and ATR as well as the Mre11/Rad50/Nbs (MRN) complex, which contains two highly conserved proteins Mre11 and Rad50 and a third less-conserved component, Nbs/Xrs2 (also known as nibrin), play critical roles in the response to DNA damage and telomere maintenance in mammalian systems. The primary function of the MRN complex is to sense DNA strand breaks and then to amplify the initial signal and convey it to downstream effectors, such as ATM, p53, Nbs1 (as a target of ATM), SMC1 and Brca1, that regulate cell cycle checkpoints and DNA repair. Mre11-Rad50 can bind DNA and that Mre11 possesses a nuclease activity that can process these ends. Nbs stimulates the DNA binding and nuclease activity by Mre11-Rad50. In vivo, Nbs is responsible for translocating the MRN complex to the nucleus and relocalizing the complex to the sites of DSBs following irradiation. The MRN complex is also required for activation of the S-phase checkpoint following DNA damage (Ciapponi, 2006).
It has been shown that mutations in the Drosophila mre11 and rad50 genes cause both telomere fusion and chromosome breakage. This study analyzed the role of the Drosophila nbs gene in telomere protection and the maintenance of chromosome integrity. Larval brain cells of nbs mutants display telomeric associations (TAs) but the frequency of these TAs is lower than in either mre11 or rad50 mutants. Consistently, Rad50 accumulates in the nuclei of wild-type cells but not in those of nbs cells, indicating that Nbs mediates transport of the Mre11/Rad50 complex in the nucleus. Moreover, epistasis analysis revealed that rad50 nbs, tefu (ATM) nbs, and mei-41 (ATR) nbs double mutants have significantly higher frequencies of TAs than either of the corresponding single mutants. This suggests that Nbs and the Mre11/Rad50 complex play partially independent roles in telomere protection and that Nbs functions in both ATR- and ATM-controlled telomere protection pathways. In contrast, analysis of chromosome breakage indicated that the three components of the MRN complex function in a single pathway for the repair of the DNA damage leading to chromosome aberrations (Ciapponi, 2006).
This study shows that the wild-type function of the Drosophila nbs gene is required to maintain chromosome integrity and to prevent telomere fusion. The results indicate that the nbs, mre11, and rad50 genes function in single pathway for the repair of spontaneous DNA lesions leading to chromosome breakage. In addition, it was found that double mutants affecting a single component of the MRN complex and either the ATM or the ATR kinase exhibit more chromosome breaks than the corresponding single mutants. The simplest interpretation of these results is that the two kinases function in multiple pathways for the repair of the DNA damage leading to chromosome breakage and that some of these pathways do not include the MRN complex. The finding that mei-4129D tefuatm6 double mutants and tefuatm6 single mutants display similar frequencies of chromosome breaks indicates that the ATM and ATR play redundant roles in the protection from spontaneous chromosome breakage. However, tefu and mei-41 mutants are four- and eightfold more sensitive than wild type to the X-ray induction of chromosome breakage, respectively. Thus, ATR may play a principal role in the repair of the lesions leading to chromosome breaks, with ATM playing a backup role (Ciapponi, 2006).
Recent work has shown that ATM and ATR/ATRIP function in different but redundant pathways of Drosophila telomere protection, with ATM playing an essential role and ATR compensating for the loss of ATM activity. This study shows that the frequencies of TAs observed in nbs tefu and rad50 nbs double mutants are significantly higher than those observed in the corresponding single mutants. An interpretation of these findings is that the Nbs protein functions in a telomere protection pathway that is different from either the ATR/ATRIP or the ATM/Rad50/Mre11 pathway. Alternatively, Nbs could function in both the ATM- and ATR-controlled pathways. These results are at odds with those obtained in budding yeast, where Tel1 (the ATM homolog), Rad50, Mre11, and Xrs2 (the NBS homolog) function in a single pathway of telomere maintenance. However, they are consistent with several results obtained in human cells, showing that the NBS and the MRE11/RAD50 components of the MRN complex can function independently. For example, it has been shown that NBS1 and the MRE11/RAD50 complex have separate roles in both ATM activation and ATM-mediated phosphorylation events. Moreover, while NBS1 localization to the human telomeres is restricted to the S phase, the MRE11/RAD50 complex remains associated with telomeres throughout the cell cycle (Ciapponi, 2006 and references therein).
The results suggest a model for the role of Nbs in Drosophila telomere protection. This model is based on the assumption that Nbs can facilitate both ATR- and ATM-mediated phosphorylation events, as recently shown in mammalian systems. It is proposed that Nbs is involved in both the Rad50/Mre11/ATM and the ATR/ATRIP telomere protection pathways. Nbs would mediate the transport of the Rad50/Mre11 complex in the nucleus in the Rad50/Mre11/ATM pathway and facilitate certain ATR-mediated phosphorylation events in the ATR/ATRIP pathway. Taking into account that the ATR/ATRIP telomere protection pathway is redundant (Bi, 2005), the model can explain the results of the epistasis analysis. It is speculated that in nbs mutants both pathways are partially impaired, resulting in a relatively low frequency of TAs. In rad50 nbs and tefu nbs double mutants, the Rad50/Mre11/ATM pathway would be disrupted and the ATR/ATRIP pathway partially impaired, resulting in TA frequencies higher than those found in the single mutants. Finally, in mei-41 tefu, mus-304 tefu, mei-41 rad50, and mei-41 mre11 double mutants, both pathways would be disrupted, resulting in very high frequencies of TAs (Ciapponi, 2006).
An aspect of the phenotype that is difficult to explain is the pattern of HOAP localization in different mutants and double mutants. In the mre11 and rad50 mutants, most mitotic telomeres are devoid of the HOAP protein. In nbs mutants, the frequency of telomeres with detectable HOAP accumulations is lower than in wild type but higher than in either the mre11 or the rad50 mutant, consistent with a reduced intranuclear concentration of the Rad50/Mre11 complex. tefu (ATM) and mei-41 (ATR) single mutants have normal HOAP concentrations at mitotic telomeres (Bi, 2004) but in mei-41 tefu double mutants telomeres lack the HOAP protein (Bi, 2005). Normal HOAP accumulations at mitotic telomeres were also found in Su(var)205 (HP1) and woc mutants that display very high frequencies of TAs, indicating that the presence of HOAP at chromosome ends is not sufficient to ensure proper telomere protection. An interpretation of these results is rather difficult, mainly because the current knowledge of the Drosophila telomere components is largely incomplete. HOAP localization at telomeres may be mediated, not only by the Rad50/Mre11 complex, but also by a factor that needs to be phosphorylated by both ATM and ATR. When this factor is not phosphorylated at its ATM-dependent site(s), telomeres are deprotected even if they accumulate normal amounts of HOAP. However, when this factor is not phosphorylated in both its ATM- and ATR-dependent sites, telomeres lose their ability to recruit HOAP. This factor cannot be HOAP itself, as recent work (Bi, 2005) has shown that the HOAP protein is not phosphorylated in a wild-type background (Ciapponi, 2006).
This study has shown that the Drosophila Nbs protein is required for transport of Rad50 in the nucleus and for prevention of telomere fusion and chromosome breakage. In addition, the results indicate that Nbs can act independently of the Rad50/Mre11 complex. Remarkably, all these features of the Drosophila Nbs protein are shared by its human counterpart. The ATLD disorder caused by hipomorphic mutations in the MRE11 gene and NBS have many overlapping features but are clinically distinct. NBS patients are characterized by microcephaly and developmental delay, while ATLD patients exhibit a mild ataxia telangiectasia-like phenotype with no microcephaly and no developmental delay. Given the functional similarities within Drosophila and human NBS proteins, it is likely that further studies on the Drosophila MRN complex will help to elucidate the molecular basis of the clinical differences between ATLD and NBS (Ciapponi, 2006 and references therein).
Analysis of terminal deletion chromosomes indicates that a sequence-independent mechanism regulates protection of Drosophila telomeres. Mutations in Drosophila DNA damage response genes such as atm/tefu, mre11, or rad50 disrupt telomere protection and localization of the telomere-associated proteins HP1 and HOAP, suggesting that recognition of chromosome ends contributes to telomere protection. However, the partial telomere protection phenotype of these mutations limits the ability to test if they act in the epigenetic telomere protection mechanism. The roles were examined of the Drosophila atm and atr-atrip DNA damage response pathways and the nbs homolog in DNA damage responses and telomere protection. As in other organisms, the atm and atr-atrip pathways act in parallel to promote telomere protection. Cells lacking both pathways exhibit severe defects in telomere protection and fail to localize the protection protein HOAP to telomeres. Drosophila nbs is required for both atm- and atr-dependent DNA damage responses and acts in these pathways during DNA repair. The telomere fusion phenotype of nbs is consistent with defects in each of these activities. Cells defective in both the atm and atr pathways were used to examine if DNA damage response pathways regulate telomere protection without affecting telomere specific sequences. In these cells, chromosome fusion sites retain telomere-specific sequences, demonstrating that loss of these sequences is not responsible for loss of protection. Furthermore, terminally deleted chromosomes also fuse in these cells, directly implicating DNA damage response pathways in the epigenetic protection of telomeres. It is proposed that recognition of chromosome ends and recruitment of HP1 and HOAP by DNA damage response proteins is essential for the epigenetic protection of Drosophila telomeres. Given the conserved roles of DNA damage response proteins in telomere function, related mechanisms may act at the telomeres of other organisms (Oikemus, 2006).
Alternative pre-mRNA splicing is a major mechanism utilized by eukaryotic organisms to expand their protein-coding capacity. To examine the role of cell signaling in regulating alternative splicing, the splicing of the Drosophila TAF1 pre-mRNA was analyzed. TAF1 encodes a subunit of TFIID, which is broadly required for RNA polymerase II transcription. TAF1 alternative splicing generates four mRNAs, TAF1-1, TAF1-2, TAF1-3, and TAF1-4, of which TAF1-2 and TAF1-4 encode proteins that directly bind DNA through AT hooks. TAF1 alternative splicing was regulated in a tissue-specific manner and in response to DNA damage induced by ionizing radiation or camptothecin. Pharmacological inhibitors and RNA interference were used to demonstrate that ionizing-radiation-induced upregulation of TAF1-3 and TAF1-4 splicing in S2 cells is mediated by the ATM (ataxia-telangiectasia mutated) DNA damage response kinase and checkpoint kinase 2 (CHK2), a known ATM substrate. Similarly, camptothecin-induced upregulation of TAF1-3 and TAF1-4 splicing is mediated by ATR (ATM-RAD3 related) and CHK1. These findings suggest that inducible TAF1 alternative splicing is a mechanism to regulate transcription in response to developmental or DNA damage signals and provide the first evidence that the ATM/CHK2 and ATR/CHK1 signaling pathways control gene expression by regulating alternative splicing (Katzenberger, 2006; Full text of article).
Small repeat-associated siRNAs (rasiRNAs) mediate silencing of retrotransposons and the Stellate locus. Mutations in the Drosophila rasiRNA pathway genes armitage and aubergine disrupt embryonic axis specification, triggering defects in microtubule polarization as well as asymmetric localization of mRNA and protein determinants in the developing oocyte. Mutations in the ATR/Chk2 DNA damage signal transduction pathway dramatically suppress these axis specification defects, but do not restore retrotransposon or Stellate silencing. Furthermore, rasiRNA pathway mutations lead to germline-specific accumulation of γ-H2Av foci characteristic of DNA damage. It is concluded that rasiRNA-based gene silencing is not required for axis specification, and that the critical developmental function for this pathway is to suppress DNA damage signaling in the germline (Klattenhoff, 2007).
Mutations in the Drosophila armi, aub, and spn-E genes disrupt oocyte microtubule organization and asymmetric localization of mRNAs and proteins that specify the posterior apole and dorsal-ventral axis of the oocyte and embryo. Mutations in these genes block homology-dependent RNA cleavage and RISC assembly in ovary lysates, RNAi-based gene silencing during early embryogenesis, rasiRNA production, and retrotransposon and Stellate silencing. Mutations in dcr-2 and ago-2 genes, by contrast, block siRNA function, but they do not disrupt the rasiRNA pathway or embryonic axis specification. The rasiRNA pathway thus appears to be required for embryonic axis specification. However, the function of rasiRNAs in the axis specification pathway has not been previously established (Klattenhoff, 2007).
Cytoskeletal polarization, morphogen localization, and eggshell patterning defects associated with armi and aub are efficiently suppressed by mnk and mei-41, which respectively encode Chk2 and ATR kinase components of the DNA damage signaling pathway. In addition, armi and aub mutants accumulate γ-H2Av foci characteristic of DNA DSBs and trigger Chk2-dependent phosphorylation of Vas, an RNA helicase required for posterior and dorsal-ventral specification. Mutations in spn-E also disrupt the rasiRNA pathway, trigger axis specification defects, and lead to germline-specific accumulation of γ-H2Av foci. Significantly, the mnk and mei-41 mutations do not suppress Stellate or HeT-A overexpression, indicating that axis specification does not directly require rasiRNA-dependent gene silencing. Based on these findings, it is concluded that the rasiRNA pathway suppresses DNA damage signaling in the female germline, and that mutations in this pathway disrupt axis specification by activating an ATR/Chk2 kinase pathway that blocks microtubule polarization and morphogen localization in the oocyte (Klattenhoff, 2007).
The cause of DNA damage signaling in armi, aub, and spn-E mutants remains to be established. In wild-type ovaries, γ-H2Av foci begin to accumulate in region 2 of the germarium, when the Spo11 nuclease (encoded by the mei-W68 gene) initiates meiotic breaks. The axis specification defects associated with DNA DSB repair mutations are efficiently suppressed by mei-W68 mutations, indicating that meiotic breaks are the source of DNA damage in these mutants. The axis specification defects and γ-H2Av focus formation associated with armi, by contrast, are not suppressed by mei-W68. mei-W68 double mutants with aub or spn-E have not been analyzed, but this observation strongly suggests that meiotic DSBs are not the source of DNA damage in rasiRNA pathway mutations. Retrotransposon silencing is disrupted in armi, aub, and spn-E mutants, and transcription of LINE retrotransposons in mammalian cells leads to DNA damage and DNA damage signaling. Loss of retrotransposon silencing could therefore directly induce the DSBs in rasiRNA pathway mutants. However, DNA damage can also lead to loss of retrotransposon silencing. Mutations in the rasiRNA pathway could therefore disrupt DNA repair and thus induce DNA damage, which, in turn, induces loss of retrotransposon silencing. Finally, the HeT-A retrotransposon is associated with telomeres, and overexpression of this element could reflect a loss of telomere protection and could damage signaling by chromosome ends in the rasiRNA pathway mutants. The available data do not distinguish between these alternatives (Klattenhoff, 2007).
In mouse, the piwi-related Argonauts Miwi and Mili bind piRNAs, 30 nt RNAs derived primarily from a single strand that appear to be related to rasiRNAs. Mutations in these genes disrupt spermatogenesis and lead to germline apoptosis, which can be induced by DNA damage signaling. Mammalian piRNAs and Drosophila rasiRNAs may therefore serve similar functions in suppressing a germline-specific DNA damage response (Klattenhoff, 2007).
The ability of a cell to sense and respond to DNA damage is essential for genome stability. An important aspect of the response is arrest of the cell cycle, presumably to allow time for repair. Ataxia telangiectasia mutated (ATM) and ATR are essential for such cell-cycle control, but some observations suggest that they also play a direct role in DNA repair. The Drosophila ortholog of ATR, MEI-41, mediates the DNA damage-dependent G2-M checkpoint. This study examined the role of MEI-41 in repair of double-strand breaks (DSBs) induced by P-element excision. mei-41 mutants were found to be defective in completing the later steps of homologous recombination repair, but have no defects in end-joining repair. It is hypothesized that these repair defects are the result of loss of checkpoint control. To test this, mitotic cyclin levels were genetically reduced and repair was examined in grp (DmChk1) and lok (DmChk2) mutants. The results suggest that a significant component of the repair defects is due to loss of MEI-41-dependent cell cycle regulation. However, this does not account for all of the defects observed. A novel role is proposed for MEI-41 in DSB repair, independent of the Chk1/Chk2-mediated checkpoint response (LaRocque, 2007; full text of article).
Meiotic checkpoints monitor chromosome status to ensure correct homologous recombination, genomic integrity, and chromosome segregation. In Drosophila, the persistent presence of double-strand DNA breaks (DSB) activates the ATR/Mei-41 checkpoint, delays progression through meiosis, and causes defects in DNA condensation of the oocyte nucleus, the karyosome. Checkpoint activation has also been linked to decreased levels of the TGFα-like molecule Gurken, which controls normal eggshell patterning. This easy-to-score eggshell phenotype was used in a germ-line mosaic screen in Drosophila to identify new genes affecting meiotic progression, DNA condensation, and Gurken signaling. One hundred eighteen new ventralizing mutants on the second chromosome fell into 17 complementation groups. This study describes the analysis of 8 complementation groups, including Kinesin heavy chain, the SR protein kinase cuaba (CG8174), the cohesin-related gene dPds5/cohiba, and the Tudor-domain gene montecristo. These findings challenge the hypothesis that checkpoint activation upon persistent DSBs is exclusively mediated by ATR/Mei-41 kinase and instead reveal a more complex network of interactions that link DSB formation, checkpoint activation, meiotic delay, DNA condensation, and Gurken protein synthesis (Barbosa, 2007),
In this study, a clonal screen was used to identify genes regulating meiotic progression in Drosophila. Instead of testing directly for defects in meiosis, an easy-to-score eggshell phenotype was used that is produced when the levels or activity of the morphogen Grk are affected. This allowed an efficient screen of a large number of mutant lines and identification of germ-line-specific genes as well as genes with essential functions. The number of new genes identified is likely less than the total number of 2R genes required for Grk synthesis and function since mutations were discarded that blocked oogenesis. Of the eight genes described in this study, five show meiotic phenotypes. dPds5, nds, and mtc delay meiotic restriction to the oocyte, although only dPds5 and nds genetically interact with mei-W68 and mei-41, respectively. trin and blv affect the morphology of the karyosome in spite of normal timing in meiotic restriction. This confirms the effectiveness of the screening method for meiotic genes. Genetic and developmental analysis of the newly identified genes provides evidence for new regulatory steps in a network that coordinates Drosophila meiosis and oocyte development (Barbosa, 2007),
One complementation group, cohiba, identifies the Drosophila homolog of Pds5p in Schizosaccharomyces pombe, Spo76 in Sordaria macrospore, and BimD in Aspergillus nidulans, which have been found associated with the cohesion complex of mitotic and meiotic chromosomes. Depletion of Pds5 affects not only cohesion but also condensation in meiotic prophase. The unique 'open chromatin' karyosome defect observed in dPds5cohiba mutants is consistent with a role of Pds5 in chromosome cohesion during Drosophila meiosis. Like Spo76, the dPds5cohiba phenotype is suppressed by Spo11 (mei-W68) mutations defective in DSB formation. This suggests that dPds5 is necessary to maintain the structure of the meiotic chromosomes after DSBs are induced. However, in contrast to known DSB repair genes, the meiotic delay and oocyte patterning defects of dPdscohiba mutants are not due to activation of ATR/Mei-41-dependent checkpoint. One possibility is that the ATR downstream effector kinase dChk2 is activated via an alternative pathway, such as the Drosophila ataxia-telangiectasia mutated (ATM) homolog, which indeed activates dChk2 in the early embryo independently of ATR. Alternatively, dPdscohiba mutants may activate a checkpoint that measures cohesion rather than DSB breaks. The only other cohesion protein characterized in Drosophila is the product of the orientation disruptor (ord). ORD plays a role in early prophase I by maintaining synaptic chromosomes and allowing interhomolog recombination. More importantly and perhaps similar to dPds5, ORD seems not to be required for DSB repair. However, in contrast to dPds5 mutants, karyosome morphology is normal in ord mutants, and an eggshell polarity phenotype has not been reported. Although required for chromatid cohesion, dPds5 and ORD might play complementary roles in SC dynamics: ORD may stabilize the SC in the oocyte, whereas dPds5 may be required for the disassembly of synapses as one of the pro-oocytes regresses from meiosis (Barbosa, 2007),
The screen identified mutations in montecristo (mtc) that affect the restriction of meiosis to the oocyte. It has been proposed that this delay reflects the activation of the ATR/Mei-41 checkpoint pathway. Similar to dPds5, Mtc may control the regression from pachytene in those cyst cells that will not adopt the oocyte fate. The delayed meiotic restriction observed in mtc mutants occurs, however, independently of DSB formation or Mei-41 checkpoint activation. Mtc contains a Tudor domain. In other Tudor-domain proteins, this domain has been shown to interact with methylated target proteins. Identification of specific Mtc targets may clarify its role in meiotic restriction and oocyte patterning (Barbosa, 2007),
A particularly intriguing and novel phenotype is uncovered by mutations in indios (nds). By delaying meiotic restriction and activating Mei-41 without affecting the karyosome morphology, nds mutants separate checkpoint activation leading to Grk decrease from checkpoint activation controlling karyosome compaction. The nds phenotype also occurs independently of DSBs, suggesting that the trigger that leads Nds to trigger checkpoint activation is not DNA breaks. The fact that nds mutants are extremely sensitive to Mei-41 dosage further suggests that Nds activity may specifically control a branch of the Mei-41 checkpoint regulating Grk activity. In contrast to nds, trin mutants do not delay meiotic restriction and show defects in the karyosome in spite of normal Grk levels. Like mutants in src64B and tec29, which show a similar phenotype, Trin may mediate chromatin remodeling in the oocyte by regulating the actin cytoskeleton. In this context, the DV phenotype of eggs from trin mutants may be an indirect effect due to defects in actin cytoskeleton function. The production of collapsed eggs by trin mutant germ-line clones is consistent with this idea (Barbosa, 2007),
Finally, blv mutants show striking similarity to vas mutants with respect to lack of sensitivity to DSB formation, no evident delays of meiotic restriction, or karyosome and Grk phenotypes. Blv may thus act downstream or independent of the Mei41/ATR checkpoint, and its further characterization may help to understand the effector side of the meiotic checkpoint pathway (Barbosa, 2007),
Previous knowledge pointed to Drosophila meiosis as a linear progression of events from homologous chromosome pairing and recombination to meiotic restriction, karyosome formation, and eggshell patterning, with DSB repair as the main checkpoint linking meiosis to Grk signaling. By uncoupling some of these events, this study suggests the existence of a more complex network that links the surveillance of meiotic progression to oocyte patterning (Barbosa, 2007).
The conserved histone variant H2A.Z fulfills many functions by being an integral part of the nucleosomes placed at specific regions of the genome. Telomeres cap natural ends of chromosomes to prevent their recognition as double-strand breaks. At yeast telomeres, H2A.Z prevents the spreading of silent chromatin into proximal euchromatin. A role for H2A.Z in capping, however, has not been reported in any organism. This study uncovered such a role for Drosophila H2A.Z. Loss of H2A.Z, through mutations in either its gene or the domino gene for the Swr1 chromatin-remodeling protein, suppressed the fusion of telomeres that lacked the protection of checkpoint proteins: ATM, ATR, and the Mre11-Rad50-NBS complex. Loss of H2A.Z partially restores the loading of the HOAP capping protein, possibly accounting for the partial restoration in capping. It is proposed that, in the absence of H2A.Z, checkpoint-defective telomeres adopt alternative structures, which are permissive for the loading of the capping machinery at Drosophila telomeres (Rong, 2008).
This study shows that loss of H2AvD in Drosophila suppresses fusion of telomeres that lack the protection of conserved checkpoint proteins: ATM, ATR, or MRN. Drosophila H2AvD encodes the functions for both H2A.X and H2A.Z variants that are translated from separate genes in other organisms. By using transgenes that either have or lack H2A.X function, it was established that H2AvD's role in regulating capping resides in its H2A.Z-homologous region. This conclusion is strengthened by the result from analyzing a domino mutation that behaved similarly to an h2AvD mutation. This represents a novel function of H2A.Z that has not been demonstrated in any other organism (Rong, 2008).
It is possible that the effect of h2AvD mutations on fusion frequencies is an indirect effect of transcriptional mis-regulation of genes controlling the repair and/or response to DSBs. This, however, is unlikely since cav mutant cells lacking the HOAP capping component are equally prone to telomere fusion with or without H2A.Z, suggesting that H2AvD mutant telomeres are not refractory to being repaired as DSBs. In addition, an h2AvD mutation was unable to suppress fusion in an atm cav h2AvD triple mutant, suggesting that cav is epistatic to h2AvD. In light of the observation that an h2AvD mutation can partially restore HOAP binding to atm atr double-mutant telomeres, it is suggested that loss of H2AvD might permit more efficient loading of capping proteins and, therefore, more efficient capping (Rong, 2008).
Another hypothesis considered is that H2AvD accumulates at checkpoint-defective telomeres, interfering with the binding of the capping machinery. However, evidence obtained from immuno-localization of H2AvD did not support this hypothesis. H2AvD has an interesting distribution on mitotic chromosomes in wild-type cells in that it is underrepresented in regions commonly considered heterochromatic. Telomeres are generally considered heterochromatic on the basis of their ability to silence nearby genes. However, recent results suggest that the heterochromatic features of Drosophila telomeres reside in the subtelomeric telomere-associated sequence (TAS) repeats and that the retro-transposon arrays at the extreme of chromosome ends possess certain euchromatic features (Biessmann, 2005). This is consistent with the fact that telomeric retro-transposons are actively transcribed to serve as transposition intermediates (Pardue, 2008). Therefore, H2AvD may not be excluded from wild-type telomeric regions, a suggestion supported by a recent genomewide localization study (Mavrich, 2008). Nevertheless, no elevated level of H2AvD was observed at checkpoint-defective telomeres even though these experiments were set up to favor detection of such enrichment. First, the atm atr double mutant - which has the strongest capping defects and on which the h2AvD mutation had the strongest suppressing effect - was included. Second, H2AvD enrichment would have been prominently detected on telomeres from the Y and fourth chromosomes as well as the short arm of the X chromosome on which H2AvD is normally underrepresented. Therefore, it is unlikely that H2AvD interferes with HOAP loading at checkpoint-defective telomeres and that the loss of such interference partly restores capping in h2AvD mutants (Rong, 2008).
Finally, the absence of H2A.Z might allow telomeres to adopt an alternative structure that is permissive to the loading of capping proteins. At S. cerevisiae telomeres, H2A.Z may demarcate the euchromatin-heterochromatin boundary. It may serve a similar function in Drosophila. Interestingly, recent results suggest that the heterochromatic features of Drosophila telomeres might reside in the subtelomeric TAS regions (Biessmann, 2005). It is possible that H2AvD prevents the spreading of TAS-associated heterochromatin into the transposon arrays. In the absence of H2AvD, Drosophila telomeres might adopt a heterochromatin-like structure, which facilitates the loading of capping proteins. This model is purely speculative due to the fact that the structure of Drosophila telomeres is poorly understood. In particular, the structural elements necessary for the loading of capping machinery remain undetermined. Nevertheless, due to the high degree of conservation in H2A.Z variants from different organisms, its role in regulating telomere capping uncovered in this study may also be conserved (Rong, 2008).
In both yeast and mammals, uncapped telomeres activate the DNA damage response (DDR) and undergo end-to-end fusion. Previous work has shown that the Drosophila HOAP protein, encoded by the caravaggio (cav) gene, is required to prevent telomeric fusions. This study shows that HOAP-depleted telomeres activate both the DDR and the spindle assembly checkpoint (SAC). The cell cycle arrest elicited by the DDR was alleviated by mutations in mei-41 (encoding ATR), mus304 (ATRIP), grp (Chk1) and rad50 but not by mutations in tefu (ATM). The SAC was partially overridden by mutations in zw10 (also known as mit(1)15) and bubR1, and also by mutations in mei-41, mus304, rad50, grp and tefu. As expected from SAC activation, the SAC proteins Zw10, Zwilch, BubR1 and Cenp-meta (Cenp-E) accumulated at the kinetochores of cav mutant cells. Notably, BubR1 also accumulated at cav mutant telomeres in a mei-41-, mus304-, rad50-, grp- and tefu-dependent manner. These results collectively suggest that recruitment of BubR1 by dysfunctional telomeres inhibits Cdc20-APC function, preventing the metaphase-to-anaphase transition (Musarò, 2008).
In most organisms, telomeres contain arrays of tandem G-rich repeats added to the chromosome ends by telomerase. Drosophila telomeres are not maintained by the activity of telomerase, but instead by the transposition of three specialized retrotransposons to the chromosome ends. In addition, whereas yeast and mammalian telomeres contain proteins that recognize telomere-specific sequences, Drosophila telomeres are epigenetically determined, sequence-independent structures. Nonetheless, Drosophila telomeres are protected from fusion events, just as their yeast and mammalian counterparts are. Genetic and molecular analyses have thus far identified eight loci that are required to prevent end-to-end fusion in Drosophila: effete (eff, also known as UbcD1), which encodes a highly conserved E2 enzyme that mediates protein ubiquitination; Su(var)205 and caravaggio (cav), encoding HP1 and HOAP, respectively; the Drosophila homologs of the ATM, RAD50, MRE11A and NBN (also known as NBS1) genes; and without children (woc), whose product is a putative transcription factor (Musarò, 2008).
To determine whether mutations in genes required for telomere capping also affect cell cycle progression, DAPI-stained preparations of larval brains from seven of these eight telomere-fusion mutants were examined. Mutant brains were examined for the mitotic index (MI) and the frequency of anaphases (AF). The mitotic indices observed for the eff, Su(var)205, mre11, rad50, woc and tefu mutants ranged from 0.46 to 0.75, values that were slightly lower than the mitotic index observed for the wild type (0.86). However, brains from cav mutants showed a fourfold reduction of the mitotic index (0.19) with respect to the wild type. cav mutants also had a very low frequency of anaphases (1.7%-1.9%) compared to the wild type (13.2%), whereas in the other mutants, frequency of anaphases ranged from 8.6% to 12.5%. Reductions in both the mitotic index and the frequency of anaphases were rescued by a cav+ transgene, indicating that these phenotypes were indeed due to a mutation in cav (Musarò, 2008).
These results prompted a focus on cav mutations in order to determine how unprotected telomeres might influence cell cycle progression. The cav allele used in this study is genetically null for the telomere-fusion phenotype. cav homozygotes and cav1/Df(3R)crb-F89-4 hemizygotes show very similar mitotic indices and frequencies of anaphases, indicating that cav is also null for these cell cycle parameters. The cav-encoded HOAP protein localizes exclusively to telomeres; cav produces a truncated form of HOAP that fails to accumulate at chromosome ends (Musarò, 2008).
The low frequencies of anaphases observed in cav mutant cells suggest that they may be arrested in metaphase. To confirm a metaphase-to-anaphase block, mitoses were filmed of cav and wild-type neuroblasts expressing the GFP-tagged H2Av histone. Control cells entered anaphase within a few minutes after chromosome alignment in metaphase, whereas cav cells remained arrested in metaphase for the duration of imaging (Musarò, 2008).
It was hypothesized that the cav-induced metaphase arrest was the result of SAC activation. As in all higher eukaryotes, unattached Drosophila kinetochores recruit three SAC protein complexes (Mad1-Mad2, Bub1-BubR1-Cenp-meta and Rod-Zw10-Zwilch) that prevent precocious sister chromatid separation by negatively regulating the ability of Cdc20 to activate the anaphase-promoting complex or cyclosome (APC/C). Mutations in genes encoding components of these complexes lead to SAC inactivation and allow cells to enter anaphase even if the checkpoint is not satisfied. To ask whether the low frequency of anaphases in cav mutant brains was due to SAC activation, zw10 cav and bubR1 cav double mutants were analyzed. In both cases, the frequency of anaphases was significantly higher than in the cav single mutant, whereas the frequency of telomere fusions remained unchanged. These results imply that the low frequency of anaphases in cav mutants is indeed due to SAC activation (Musarò, 2008).
SAC activation would be expected to increase the mitotic index through the accumulation of metaphase cells; however, in cav single mutants, the mitotic index is abnormally low. One explanation for this apparent paradox is that the cell cycle in cav cells is also delayed before M-phase, as a result of the DNA damage response (DDR). To ask whether HOAP-depleted telomeres activate any DNA damage checkpoints, double mutants were generated for cav and genes known to be involved in these checkpoints: mei-41 and telomere fusion (tefu), encoding the fly homologs of ATR and ATM, respectively; mus304, which encodes the ATR-interacting protein ATRIP grapes (grp), which specifies a CHK1 homolog and rad50, whose product is part of the Mre11-Rad50-Nbs complex. DAPI-stained preparations of larval brain cells from these double mutants showed that mei-41, mus304, grp and rad50 mutations alleviate the cell cycle block induced by cav, causing a ~2.5-fold increase of the mitotic index relative to that observed in the cav single mutant. In contrast, the tefu mutation did not affect the cav- induced interphase block. These effects are unrelated to variations in the frequency of telomere fusions, as the telomere fusion frequencies in double mutants were very similar to those in the cav single mutant. It is thus concluded that the interphase arrest in cav mutants occurs independently of ATM and is mediated by a signaling pathway involving ATR, ATRIP, Chk1 and Rad50. This signaling pathway is known to activate DNA damage checkpoints during the G1/S transition, the S phase and the G2/M transition. However, the current results do not allow identification of the particular checkpoint(s) activated by HOAP-depleted telomeres (Musarò, 2008).
Notably, in all double mutants for cav and any one of the genes associated with the DDR, including tefu (ATM), a significant increase was also observed in the frequencies of anaphases relative to that of the cav single mutant, suggesting that these genes are involved in the cav-induced metaphase arrest. This finding reflects a role of these DDR-associated genes in the peculiar mechanism by which uncapped Drosophila telomeres activate SAC (Musarò, 2008).
To obtain further insight about the cav-induced metaphase arrest, the localization of Zw10, Zwilch, BubR1 and Cenp-meta (Cenp-E) was determined by immunofluorescence. In wild-type Drosophila cells, these proteins begin to accumulate at kinetochores during late prophase and remain associated with kinetochores until the chromosomes are stably aligned at the metaphase plate. Treatments with spindle poisons (for example, colchicine) disrupt microtubule attachment to the kinetochores, leading to metaphase arrest with SAC proteins accumulated at the centromeres. Immunostaining for Zwilch, Zw10, Cenp-meta or BubR1 showed that in all cases, the frequencies of cav metaphases with strong centromeric signals were comparable to those observed in colchicine-treated wild-type cells, and they were significantly higher than those seen in untreated wild-type metaphases. These findings support the view that HOAP-depleted telomeres activate the canonical SAC pathway (Musarò, 2008).
Through a detailed examination of cav metaphases immunostained for SAC proteins, an unexpected connection was found between uncapped telomeres and the localization of at least one SAC component. Although Zwilch, Zw10 and Cenp-meta accumulated exclusively at kinetochores, BubR1 was concentrated at both kinetochores and telomeres. BubR1 localized at both unfused (free) and fused telomeres; most (94.4%) cav metaphases showed at least one telomeric BubR1 signal. To better resolve the chromosome tangles seen in cav metaphases, cells were treated with hypotonic solution, allowing a focus on free telomeres, which can be reliably scored. It was found that 25% of the free telomeres in cav metaphases show an unambiguous BubR1 signal. BubR1 accumulations were not observed at wild-type telomeres or at the breakpoints of X-ray-induced chromosome breaks. BubR1 localization at telomeres was not caused by the formation of ectopic kinetochores at the chromosome ends, since cav telomeres did not recruit the centromere and kinetochore marker Cenp-C. Low frequencies of BubR1-labeled telomeres were also observed in other mutant strains with telomere fusions including eff, Su(var)205 and woc. These results indicate that BubR1 specifically localizes at uncapped telomeres (Musarò, 2008).
It was next asked whether mutations in mei-41, grp, mus304, tefu, rad50 and zw10 affect BubR1 localization at cav mutant telomeres. Whereas mutations in zw10 did not affect BubR1 localization at cav chromosome ends, double mutants for cav and any of the other genes all showed significant reductions in the frequency of BubR1-labeled free telomeres with respect to cav single mutants. Considered together, these results indicate that when the canonical SAC machinery is intact (in all cases except in zw10 cav double mutants), there is a strong negative correlation between the frequency of BubR1-labeled telomeres and the frequency of anaphases. These findings suggest that BubR1 accumulation at telomeres can activate the SAC (Musarò, 2008).
Finally it was asked whether mutations in DDR-associated genes can allow cells to bypass the SAC when it is activated by spindle abnormalities rather than by uncapped telomeres. The spindle was disrupted in two ways: with the microtubule poison colchicine and with mutations in abnormal spindle (asp). Both situations activated the SAC and caused metaphase arrest; neither mei-41 nor grp or tefu mutations allowed cells to bypass this arrest, whereas mutations in zw10 led such cells to exit mitosis. These findings indicate that the DDR-associated genes regulate BubR1 accumulation at cav telomeres but are not directly involved in the SAC machinery (Musarò, 2008).
Collectively, these results suggest a model for the activation of cell cycle checkpoints by unprotected Drosophila telomeres. It is proposed that uncapped telomeres activate DDR checkpoints, leading to interphase arrest through a signaling pathway involving mei-41 (encoding ATR), mus304 (ATRIP), grp (Chk1) and rad50, but not tefu (ATM). This pathway is independent of telomeric BubR1, because mutations in tefu, which strongly reduce BubR1 accumulation at chromosome ends, do not rescue cav-induced interphase arrest. Uncapped telomeres can also activate the SAC by recruiting BubR1 through a pathway requiring mei-41, mus304, grp, rad50 and tefu functions. Once accumulated at the telomeres, BubR1 may negatively regulate either Fizzy (Cdc20) or another APC/C subunit so as to cause metaphase arrest. This model posits that certain DDR-associated genes, such as rad50, function both in the DDR pathway and in the pathway that mediates BubR1 recruitment at telomeres. This explains why rad50 and mre11 mutants show only mild reductions of the mitotic index and the frequency of anaphases even though HOAP is substantially depleted from their telomeres (Musarò, 2008).
It is proposed that uncapped telomeres can induce an interphase arrest independently of BubR1 through a signaling pathway that involves ATR, ATRIP, CHK1 and Rad50 but not ATM. The same proteins, including ATM, are required for the recruitment of BubR1 at unprotected telomeres. Telomeric BubR1 may negatively regulate the activity of the Cdc20-APC complex, leading to a metaphase-to-anaphase transition block. The metaphase arrest caused by Cdc20-APC inhibition is likely to cause an accumulation of SAC proteins on the kinetochores, reinforcing SAC activity. Consistent with this view, mutations in ida, which encodes an APC/C subunit, lead to a metaphase arrest phenotype with BubR1 accumulated at the kinetochores (Musarò, 2008).
Several recent reports have suggested possible relationships between DNA damage, SAC and telomeres. In both Drosophila and mammalian cells, DNA breaks can activate the SAC, presumably by disrupting kinetochore function. In Schizosaccharomyces pombe, Taz1-depleted telomeres result in Mph1p- and Bub1p-mediated SAC activation, and mutations in yKu70 affecting Saccharomyces cerevisiae telomere structure also activate the SAC. However, these previous studies did not explain how telomere perturbations might be perceived by the SAC. This study has found that unprotected Drosophila telomeres recruit the BubR1 kinase as do the kinetochores that are unconnected to spindle microtubules. Thus, it is possible that telomere-associated BubR1 inhibits anaphase through molecular mechanisms similar to those that govern SAC function at the kinetochore. Consistent with this possibility, a single BubR1 accumulation at either a centromere or a telomere seems competent to block anaphase onset. It will be of interest in the future to establish whether deprotected mammalian telomeres can also activate the SAC and, if so, whether BubR1 recruitment to the damaged telomeres mediates this response (Musarò, 2008).
ATR and Chk1 are protein kinases that perform major roles in the DNA replication checkpoint that delays entry into mitosis in response to DNA replication stress by hydroxyurea (HU) treatment. They are also activated by ionizing radiation (IR) that induces DNA double-strand breaks. Studies in human tissue culture and Xenopus egg extracts identified Claspin as a mediator that increased the activity of ATR toward Chk1. Because the in vivo functions of Claspin are not known, Drosophila lines were generated that each contained a mutated Claspin gene. Similar to the Drosophila mei-41/ATR and grp/Chk1 mutants, embryos of the Claspin mutant showed defects in checkpoint activation, which normally occurs in early embryogenesis in response to incomplete DNA replication. Additionally, Claspin mutant larvae were defective in G2 arrest after HU treatment; however, the defects were less severe than those of the mei-41/ATR and grp/Chk1 mutants. In contrast, IR-induced G2 arrest, which was severely defective in mei-41/ATR and grp/Chk1 mutants, occurred normally in the Claspin mutant. It was also found that Claspin is phosphorylated in response to HU and IR treatment and a hyperphosphorylated form of Claspin is generated only after HU treatment in mei-41/ATR-dependent and tefu/ATM-independent way. In summary, these data suggest that Drosophila Claspin is required for the G2 arrest that is induced by DNA replication stress but not by DNA double-strand breaks, and this difference is probably due to distinct phosphorylation statuses (Lee, 2012).
Claspin was originally identified in Xenopus laevis egg extracts as a Chk1-interacting protein that was required for DNA replication stress-induced G2 arrest. DNA replication stress induces ATR-dependent phosphorylation of Claspin, which results in a Claspin-Chk1 interaction and phosphorylation and activation of Chk1 by ATR. Claspin protein levels are regulated during the cell cycle, peak at the S/G2 boundary, and are degraded during mitosis. In support of its high expression during S phase, Claspin is reported to have a role during normal DNA replication when in the absence of exogenous DNA damage. Moreover, Claspin is required for terminating DNA damage-induced cell cycle arrest. Phosphorylation of Claspin by Polo-like kinase-1 (Plk1) results in the dissociation of Claspin from chromatin in Xenopus or the degradation of Claspin in human cells, which leads to inactivation of Chk1 and resumption of the cell cycle after prolonged interphase arrest. Most of the work on Claspin has been performed in Xenopus egg extracts and human tissue culture, however, animal models for Claspin have not been reported (Lee, 2012).
To understand the in vivo function of Claspin, mutants of Claspin (CG32251) were generated by imprecise excision of a transposable element. Analysis of the DNA damage checkpoint during early embryogenesis and the larval stage of this mutant showed that Drosophila Claspin is required for cell cycle arrest in response to incompletely replicated DNA. However, Claspin is dispensable for IR-induced cell cycle arrest. Interestingly, Claspin is phosphorylated after IR and HU treatment and a hyperphosphorylated form of Claspin was observed after HU but not after IR treatment. Moreover, the HU-induced hyperphosphorylation of Claspin is attenuated in mei-41/ATR mutant, but not in tefu/ATM mutant. These results suggest that the phosphorylation state and the role of Drosophila Claspin in cell cycle arrest are distinctly regulated by different types of DNA damage: DNA replication stress and DSBs (Lee, 2012).
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date revised: 22 April 2021
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