Chk2/Dmnk (Drosophila maternal nuclear kinase) is a Drosophila gene encoding a putative nuclear protein serine/threonine kinase with no apparent homology to previously identified protein kinases and located at 38B on the second chromosome. Dmnk mRNAs are transcribed in nurse cells and are subsequently localized in the anterior of oocytes during oogenesis, in a manner similar to several maternal transcripts regulating oogenesis and early embryogenesis. At early cleavage-stages Dmnk transcripts are transiently present throughout the embryo, but become restricted to the posterior pole and then to the newly-formed primordial germ cells (pole cells) by the blastoderm stage. The transcripts are sustained in the pole cells during gastrulation until they pass through the midgut pocket wall into the body cavity. Immunostaining with specific antibodies reveals that Dmnk proteins are localized to the nuclei in a speckled pattern. Dmnk proteins become detectable in both somatic and germ line cell nuclei upon their arrival at the periplasm of the syncytial embryo, but then disappear from the somatic cell nuclei. Consistent with mRNA expression, Dmnk proteins in pole cell nuclei are sustained during gastrulation. When taken together, Dmnk appears to represent a novel class of nuclear protein kinases and the dynamic expression of Dmnk suggests a role in germ line establishment (Oishi, 1998).
Northern hybridization with the chk2 cDNA reveals that the expression of its RNA is temporally restricted during development. The chk2 RNA appears to accumulate primarily in the ovary and in embryos during the first 2 hr of development, indicating that chk2 RNA is maternally deposited into the egg. Although the chk2 transcript signal is not spatially restricted to any part of the young embryos (Oishi, 1998), in ovaries, chk2 mRNA is first detected in region 2 of the germarium, where it concentrates in the prospective oocyte. Later it is clearly found to accumulate in the oocyte, at the posterior end up to stage 7. From stage 8 to 10, it is found at the anterior cortex of the oocyte (Masrouha, 2003).
A polyclonal antibody raised against Chk2 recognizes a specific band of ~52 kD in wild-type embryos but not in embryos from chk2 mutant mothers. Even though chk2 mRNA accumulates at high levels during oogenesis, the protein is hardly detectable in this tissue. However, a strong Chk2 signal is found during the first 4 hr of embryonic stages (04 hr after egg deposition). After that, the Chk2 signal drops again to background levels (Masrouha, 2003).
Chk2 is a major target of ataxia telangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR). Germline mutations in Chk2 have been identified in a subset of patients with Li-Fraumeni syndrome, suggesting that Chk2 is a tumor suppressor gene. To investigate the role of Chk2 in multicellular organisms, a Drosophila chk2 mutant was generated. chk2 mutants are viable but show defects in maintaining genome stability and are highly sensitive to ionizing radiation. Interestingly, mutating chk2 completely blocks DNA damage-induced apoptosis and partially blocks DNA damage-induced cell cycle arrest. These results indicate that Chk2 protein plays a crucial role in the DNA damage response pathway mediating cell cycle arrest and apoptosis, and that the ATM-Chk2 pathway is likely conserved in Drosophila (Xu, 2001).
Imprecise excision of the P element that inserted near chk2 was carried out. A total of 164 excision lines were established, among which 93 lines appear to be precise excision by Southern analysis. These lines were viable and showed no deletions of the chk2 coding sequences. All the remaining lines were homozygous lethal. Four lines were identified that appear to have deletion of the chk2 coding sequence by Southern blot analysis. Insertion of a P element between chk2 and barren leads to the appearance of a 3.6 kb fragment. In the E51 line, this 3.6 kb fragment is shifted to 2.0 kb, suggesting the deletion of sequences near chk2 took place. Sequencing analysis revealed that the deletion in line E51 removes the start of the open reading frame as well as all of the promoter sequences of both chk2 and barren genes. To generate flies with mutation in chk2 but not barren, a barren genomic rescue construct was created. Introduction of the barren rescue construct completely rescues the lethality of the E51 deletion line, indicating that the lethality of the E51 line observed is due to deletion of barren function. Consistent with the idea that the rescued E51 line is a mutant for chk2, no expression of chk2 was detected in rescued female while the chk2 message was abundant in wild-type control. In summary, these results show that the barren-rescued E51 line is an allele of chk2 mutant (Xu, 2001).
Studies of the C. elegans Chk2 homolog, Cechk2, suggest that Cechk2 is essential for meiosis but dispensable for DNA damage checkpoint control (Oishi, 2001; Higashitani, 2000; MacQueen, 2001). To determine if Drosophila Chk2 may have a function in DNA damage checkpoint control, radiation sensitivity of chk2 mutant larvae was compared to chk2 heterozygotes. Treatment of chk2 mutant larvae leads to a significantly higher number of larvae or pupae lethality compared to chk2 heterozygote. This result suggests that chk2 mutants are more sensitive to ionizing radiation. In addition, tests were performed to see if chk2 mutants show increased genome instability using the loss of heterozygosity (LOH) assay. As expected, mutation of chk2 leads to a much higher LOH rate. Furthermore, the rate of LOH is increased upon low dose (3 Gy) IR challenge. These phenotypes of chk2 mutants suggest that Drosophila Chk2 has an important role in maintaining genome stability and in DNA damage checkpoint response (Xu, 2001).
During Drosophila development, DNA damage leads to increased cell death and cell cycle arrest in wing discs. To further characterize the role of Chk2 in the response to DNA damage, the effect of the chk2 mutation on apoptosis and cell cycle arrest was tested. Apoptotic cells can be visualized by the dye acridine orange (AO). In wild-type wing discs, DNA damage leads to significant increase in the amount of apoptosis. In contrast, no significant increase in apoptosis was observed in chk2 mutants upon irradiation. To demonstrate that the observed block of increased apoptosis is due to lack of Drosophila chk2 function, Chk2 protein was expressed in the chk2 mutant background upon irradiation. Expression of Chk2 rescues the apoptosis block in chk2 mutants after irradiation. These results indicate that the Chk2 protein is required for radiation-induced apoptosis in the fly. It has been shown that expression of Drosophila dominant negative p53 in the wing discs also blocks radiation-induced apoptosis. Thus these observations are suggestive that Drosophila Chk2 functions in the same pathway as p53 in radiation-induced apoptosis. These results, in conjunction with the observations in mammalian systems that chk2 null ES cells exhibit resistance to apoptosis in response to DNA damage due to loss of p53 activation, suggest the Chk2-p53 pathway is likely conserved between Drosophila and mammals in response to DNA damage (Xu, 2001).
To examine the effects of chk2 mutation on radiation-induced cell cycle arrest, the ability of cells to enter mitosis after irradiation was examined. Wild-type wing disc cells are arrested after irradiation. In contrast, wing discs from mei-41 mutant larvae do not show cell cycle block, since there is a similar number of cells in M phase as in non-irradiated wing discs. Interestingly, while there are significant numbers of cells that enter mitosis upon irradiation of chk2 mutant wing discs, the numbers are much fewer than those observed in mei-41 discs or in unirradiated chk2 discs. These results indicate that Chk2 protein is required for complete cell cycle arrest following DNA damage. To demonstrate the observed defect in cell cycle arrest is due to lack of Chk2 function, rescue experiments were carried out to test if expression of Chk2 can restore the cell cycle arrest upon irradiation. Expression of Chk2 in the chk2 mutant background rescues the bypass of radiation-induced cell cycle arrest in chk2 mutants (Xu, 2001).
A null mutation of the Drosophila chk1 homolog grp also has a partial checkpoint defect, similar to the partial DNA damage-induced cell cycle arrest found in chk2 mutants. In contrast, mei-41 mutants show a complete defect in cell cycle check arrest after DNA damage. These observations are consistent with the idea that chk2 and grp both function downstream of mei-41 to bring about the cell cycle arrest. In contrast to the effect of chk2 in radiation-induced apoptosis, it was found that the grp mutant has no effect in IR-induced cell death, suggesting that Chk2 is the only target of Mei-41 that mediates radiation-induced apoptosis, while both chk2 and grp function in the DNA damage-induced cell cycle arrest pathway (Xu, 2001).
In syncytial Drosophila embryos, damaged or incompletely replicated DNA triggers centrosome disruption in mitosis, leading to defects in spindle assembly and anaphase chromosome segregation. The damaged nuclei drop from the cortex and are not incorporated into the cells that form the embryo proper. A null mutation in the Drosophila checkpoint kinase 2 tumor suppressor homolog (Chk2) blocks this mitotic response to DNA lesions and also prevents loss of defective nuclei from the cortex. In addition, DNA damage leads to increased Chk2 localization to the centrosome and spindle microtubules. Chk2 is therefore essential for a 'mitotic catastrophe' signal that disrupts centrosome function in response to genotoxic stress and ensures that mutant and aneuploid nuclei are eliminated from the embryonic precursor pool (Takada, 2003).
In syncytial embryos, replication checkpoint mutations, UV light, X-rays, and the replication inhibitor aphidicolin induce cytologically identical mitosis-specific centrosome defects and anaphase chromosome segregation failures. To further characterize the types of DNA lesions that trigger mitosis-specific centrosome inactivation and mitotic failures, spindle assembly and microtubule nucleation was directly assayed in embryos treated with another DNA replication inhibitor and a variety of DNA damaging agents. For these studies, pharmacological agents were coinjected with rhodamine-conjugated tubulin and time-lapse confocal microscopy was used to directly assay centrosome activity and spindle assembly (Takada, 2003).
Aphidicolin inhibits replication by directly interacting with DNA polymerase α, and embryos treated with this agent show mitosis-specific centrosome inactivation. To determine if this is a secondary response to aphidicolin or a consequence of the replication block, embryos were injected with the DNA replication inhibitor hydroxyurea (HU), which disrupts synthesis of deoxyribonucleotides. HU, like aphidicolin, does not visibly alter centrosome function through interphase, but consistently leads to mitosis-specific defects in centrosome function and anastral spindles assembled. As observed with aphidicolin, mitosis is prolonged, anaphase chromosome segregation fails, and centrosome function is restored during late anaphase and telophase. These observations support the conclusion that incomplete DNA replication at the onset of mitosis triggers centrosome inactivation and division failures (Takada, 2003).
Centrosome function was examined in embryos treated with drugs that cause DNA double-strand breaks. The topoisomerase I inhibitor camptothecin and the topoisomerase II inhibitors etoposide and VM-26 produce DNA strand breaks by inhibiting DNA religation. All three drugs trigger a strong centrosome inactivation response, mitotic delays, and segregation failures. Furthermore, the majority of nuclei are lost from the cortical monolayer following division failure, leaving the centrosomes behind. Embryos were also injected with the topoisomerase II inhibitor ICRF193, which does not directly cause DNA double-strand breaks during interphase, but disrupts chromosome condensation and segregation. In contrast to VM26, camptothecin and etoposide, ICRF193 did not trigger centrosome inactivation or mitotic delays. However, the chromosomes fail to segregate and the resulting abnormal nuclei drop into the interior of the embryo, leaving the centrosomes behind (Takada, 2003).
These observations indicate that defects in chromosome condensation due to inhibition of topoisomerase are insufficient to trigger centrosome inactivation or mitotic delays. Instead, it was speculated that the DNA damage produced by topoisomerase inhibitors triggers centrosome inactivation and subsequent mitotic defects. To further test this possibility, microtubule organization was assayed following injection of bleomycin, an X-ray mimetic drug that produces DNA double-strand breaks. This agent produces a particularly strong and consistent centrosome inactivation response, with 100% of injected embryos showing mitosis-specific centrosome defects. Typically, centrosomes associated with all of the nuclei in the field of view showed these defects, and essentially all of the defective nuclei produced by division failure were lost from the cortex. Embryos were also injected with restriction-digested, intact circular, or single-stranded DNA. This study also suggested that DNA breaks directly trigger mitosis-specific centrosome inactivation (Takada, 2003).
During the syncytial blastoderm stage, the majority of nuclei share a common cytoplasm and injected proteins rapidly diffuse throughout the embryo. To determine if the signal triggering centrosome inactivation diffuses through the cytoplasm, microtubule reorganization was assayed following induction of DNA damage in a single nucleus within the syncytium. For these studies, embryos were injected with rhodamine-tubulin and the DNA dye Oli-green and the confocal laser was used to irradiate a single nucleus with intense 488 nm light, which excites Oli-green and causes local photodamage. The irradiated nucleus and surrounding nuclei were then monitored as the embryo progressed into mitosis. The inactivating signal appears to be restricted to centrosomes in direct contact with damaged nuclei (Takada, 2003).
Thus essentially identical defects to those seen in checkpoint mutant embryos are observed when wild-type embryos are forced to initiate mitosis in the presence of DNA damage or incomplete replication, indicating that centrosome inactivation/mitotic failure is a normal mitosis-specific response to genotoxic lesions. Following division failure, the defective nuclei drop into the interior of the embryo, while centrosomes remain associated with the cortex. Only nuclei present at the cortex following the final syncytial division are incorporated into the blastoderm. As a result, this DNA damage response prevents transmission of defective nuclei to the embryonic precursor pool (Takada, 2003).
Chk2 has an essential and apparently non-redundant function in this mitotic response to genotoxic stress. A null mutation in the mnk gene, which encodes Chk2, prevents spontaneous centrosome inactivation in checkpoint defective grp mutants and centrosome inactivation in response to a wide range of DNA damaging agents and direct injection of restriction digested DNA. Direct analysis of chromosome behavior also demonstrates that the mnk mutation suppresses the defects in anaphase chromosome movement associated with this DNA damage response. For example, in wild-type embryos treated with the DNA damaging drug bleomycin, almost all anaphase movement is blocked and the damaged chromosomes simply decondense on mitotic exit, forming a single nucleus. In mnk mutants, by contrast, bleomycin does not visibly alter spindle morphology and the damaged chromosomes show extensive anaphase movement and are often stretched between the spindle poles, producing daughter nuclei that remain linked by chromatin bridges (Takada, 2003).
Significantly, failed division in wild-type is invariably followed by loss of defective nuclei from the cortex, while defective nuclei are always retained at the cortex in mnk mutants. In wild-type embryos, the topoisomerase II inhibitor ICRF-193 does not directly induce DNA damage during interphase and does not trigger centrosome defects or anastral spindle assembly during metaphase. Nonetheless, ICRF-193 blocks mitotic chromosome condensation and segregation in cultured cells and Drosophila embryos. In Drosophila embryos, the resulting abnormal nuclei invariably drop into the interior of the embryo, leaving the centrosomes at the cortex. Nuclear loss following division failure is therefore independent of the centrosome inactivation, and does not appear to be a secondary consequence of earlier defects in spindle function. The mnk null mutation completely suppresses nuclear loss following ICRF-193 treatment, indicating that Chk2 is required for this response. Recently, it has also been shown that ICRF-193 causes DNA double-strand breaks during mitosis. Chk2-dependent nuclear loss could therefore be a response to DNA damage during mitosis (Takada, 2003).
γTuRC localization was examined following DNA damage. Wild-type embryos treated with DNA damaging agent camptothecin show mitosis-specific loss of γ-tubulin from the centrosomes and increased labeling over the anastral central spindle microtubules. Note that this response is observed in both the syncytial embryo and the pole cells, which are germline precursors and the first true cells to form during embryogenesis. Centrosome inactivation, therefore, is not restricted to the syncytial divisions. In mnk embryos, camptothecin has no effect on γ-tubulin localization, which accumulated at centrosomes in both the syncytium and pole cells. However, in mnk mutants carrying a wild-type mnk transgene, camptothecin induces wild-type loss of γ-tubulin localization in both the syncytium and pole cells. Based on these observations, it is conclude that Drosophila Chk2 is essential to centrosome inactivation in response to replication defects and a wide range of genotoxic lesions (Takada, 2003).
Based on these observations, it is proposed that Chk2 functions at two points during the early embryonic response to genotoxic stress. At the onset of mitosis, the presence of DNA lesions leads to Chk2 activation, which targets proteins involved in maintaining γ-tubulin ring complex (γTuRC) localization and centrosomal microtubule nucleating activity. The resulting anastral spindle is functionally compromised and anaphase chromosome segregation fails. Following division failure, Chk2 is required for a second process that appears to disrupt the link between centrosomes and nuclei, or prevent reestablishment of this link. Because the centrosomes anchor nuclei at the cortex, this Chk2-dependent response to DNA damage leads to loss of nuclei from the cortical monolayer. Only cortical nuclei are incorporated into the cells that will form the blastoderm embryo. The two-step Chk2-dependent response to genotoxic stress thus blocks propagation of abnormal nuclei and prevents their transmission to the embryo proper (Takada, 2003).
In Drosophila larvae and other systems, Chk2 appears to function upstream of p53 during DNA damage induced apoptosis. Because the p53 gene has been implicated in control of centrosome duplication, it is speculated that Chk2 functions through p53 during this mitosis-specific response to DNA damage. However, p53 null mutant embryos show a wild-type centrosome inactivation response. Localization of the γTuRC is disrupted during centrosome inactivation, and localization of Chk2 to the centrosome in response to DNA damage and in checkpoint defective embryos raises the possibility that this conserved kinase directly modifies one or more components of this complex. However, nuclear loss following division failure also requires Chk2, and this response is not dependent on loss of microtubule nucleating function during mitosis. Chk2 thus appears to target distinct factors involved in maintaining or establishing the link between nuclei and centrosomes. Mutations in cytoplasmic dynein lead to centrosome detachment from the nucleus during the syncytial divisions, suggesting that Chk2 could modify dynein or a dynein binding protein on mitotic exit in response to DNA damage. Identification of the downstream targets of Chk2 on mitotic onset and exit should shed light on both the mitotic DNA damage response and normal centrosome function (Takada, 2003).
Is DNA damage and replication dependent centrosome inactivation conserved? Mammalian cells do not show centrosomal defects when DNA damage is induced during mitosis, suggesting that these cells may not mount a centrosome inactivation response. However, in Drosophila embryos, centrosomal defects are not observed when DNA damage is induced after the S to M transition. It is therefore speculated that, in both flies and mammals, Chk2 must be activated during interphase in order to trigger centrosome defects on progression into mitosis. Directly supporting this hypothesis, an in vivo study examined checkpoint compromised CHO cells as they progressed into mitosis following a replication block. In these cells, the centrosomes appear cytologically normal through G2, but often appear fragmented as the cell progresses into mitosis. DNA repair deficient cells spontaneously show identical mitosis-specific centrosomal defects. Furthermore, human colon carcinoma cells treated with ionizing radiation progress though a failed mitotic division, producing single tetraploid daughters. Finally, mammalian Chk2 has recently been shown to localize to centrosomes and the midbody. These findings suggest that mammalian cells also regulate centrosome organization and spindle function in response to replication defects and DNA damage (Takada, 2003).
By coupling centrosome function and spindle assembly to genome integrity, the Chk2-dependent pathway described here maintains genome stability by blocking the propagation of mutant or aneuploid nuclei and preventing their transmission to the embryonic precursor pool. This response is therefore functionally similar to apoptosis, although the process is initiated in mitosis rather than interphase. Mutations in human Chk2 increase genomic instability and are linked to cancer. The established functions for Chk2 in checkpoint control and apoptosis almost certainly contribute to this. However, if mammalian Chk2 also couples assembly of the chromosome segregation machinery to genome integrity, loss of this mitotic function could directly increase genome instability and contribute to tumor progression in humans (Takada, 2003).
Two conflicting studies reach opposite conclusions regarding the role of chk2 as an essential element of the meiotic checkpoint in Drosophila (Abdu, 2002 and Masrouha, 2003). Both studies are reviewed in this section.
During Drosophila oogenesis, unrepaired double-strand DNA breaks activate a mei-41-dependent meiotic checkpoint, which couples the progression through meiosis to specific developmental processes. This checkpoint affects the accumulation of Gurken protein, a transforming growth factor alpha-like signaling molecule, as well as the morphology of the oocyte nucleus. However, the components of this checkpoint in flies have not been completely elucidated. A mutation in the Drosophila Chk2 homolog (DmChk2/Mnk) has been shown to suppress the defects in the translation of gurken mRNA and also the defects in oocyte nuclear morphology. Drosophila Chk2 is phosphorylated in a mei-41-dependent pathway. Analysis of the meiotic cell cycle progression shows that the Drosophila Chk2 homolog is not required during early meiotic prophase, as has been observed for Chk2 in C. elegans. The activation of the meiotic checkpoint affects Wee localization and is associated with Chk2-dependent posttranslational modification of Wee. It is suggested that Wee has a role in the meiotic checkpoint that regulates the meiotic cell cycle, but not the translation of gurken mRNA. In addition, p53 and mus304, the Drosophila ATR-IP homolog, are not required for the patterning defects caused by the meiotic DNA repair mutations. It is concluded that Chk2 is a transducer of the meiotic checkpoint in flies that is activated by unrepaired double-strand DNA breaks. Activation of Chk2 in this specific checkpoint affects a cell cycle regulator as well as mRNA translation (Abdu, 2002).
Mutations in the spindle class of double-strand break (DSB) DNA repair enzymes, such as spn-B (DMC/RAD51-like) and okr (Dmrad54), affect meiosis and dorsal-ventral patterning in Drosophila oogenesis. These mutations activate a mei-41-dependent meiotic checkpoint. mei-41 encodes a member of the ATM/ATR subfamily of phosphatidylinositol-3-OH-kinase-like proteins. Activation of the mei-41-dependent checkpoint prevents efficient translation of gurken (grk) mRNA, which results in a ventralization of eggs and embryos. The effect on grk is presumably mediated through posttranslational modification of Vasa, an eIF4A-like translation initiation factor. In addition, the oocyte nuclear morphology is abnormal in the spindle mutants. In flies homozygous for mutations in a DSB repair enzyme and in mei-41, the patterning and the oocyte nuclear defects are suppressed, demonstrating that a defect in the nucleus affects Vasa and Grk in the cytoplasm via activity of mei-41. However, how activation of mei-41 causes patterning defects is not fully understood (Abdu, 2002).
Activation of a meiotic checkpoint in response to the persistence of unrepaired DSBs appears to be a conserved regulatory feature common to yeast, worms, flies, and vertebrates. In Caenorhabditis elegans, and in mice, activation of a meiotic checkpoint leads to meiotic arrest followed by apoptosis. However, the mechanism of this specific checkpoint in these species has not been completely elucidated. In budding yeast, activation of the meiotic checkpoint inhibits cell cycle progression by limiting the abundance or activity of the components of the cyclin-dependent kinase complex (Abdu, 2002 and references therein).
The DNA damage response system in mitotic cells involves a signal transduction pathway consisting of sensors, transducers, and effectors. The identities of the sensors are unclear. The signal transducers are comprised of four sets of conserved proteins. One family is composed of ATM and ATM-Rad3-related (ATR) proteins. Downstream of these proteins are two families of checkpoint kinases, the Chk1 and the Chk2 kinases and their homologs. The fourth conserved family is that of the BRCT repeat-containing proteins (Abdu, 2002).
The term meiotic checkpoint is used in this context to refer to the regulation of Drosophila oocyte patterning and nuclear morphology in the presence of unrepaired DSBs. To further characterize the meiotic checkpoint in Drosophila, the role of several genes required for the DNA damage checkpoint was examined: the ATR-interacting protein homolog, mus304; the ATR-downstream kinases grp (Chk1) and Chk2/mnk; and the effector proteins Wee and p53. The ability of these genes to suppress the patterning and nuclear morphology defects of loss-of-function alleles in double-strand DNA repair enzymes was studied (Abdu, 2002).
Are proteins involved in mitotic DNA damage checkpoint activity also required in the meiotic checkpoint in Drosophila oogenesis? In Drosophila, mus304 encodes a coiled-coil domain protein with sequence similarity to ATR-I. mus304 is required for meiotic recombination, embryonic development, and DNA damage-induced arrest. The mus304 meiotic recombination and segregation phenotypes are similar to those reported for mei-41, suggesting that they act in a common pathway that couples progress of recombination to the meiotic cell cycle. To test whether mus304 is required in the meiotic checkpoint, double-mutant flies for mus304 and spn-B or okra were generated. Surprisingly, mus304 is not required in the meiotic checkpoint since mutations in mus304 are not able to suppress the dorsal-ventral patterning defects or the oocyte nuclear morphology defect caused by mutations in the spindle-class genes. This is the first function described for mei-41 that is not shared by mus304, and it may indicate that a low level of mei-41 activity is present in mus304 mutants and is sufficient for meiotic checkpoint function (Abdu, 2002).
One of the downstream targets of ATM/ATR during checkpoint control is Chk1 kinase. In Drosophila, grapes (grp) encodes a Chk1 homolog, which regulates the DNA replication/damage checkpoint during the late syncytial divisions. grp and mei-41 are components of a conserved DNA replication/damage checkpoint pathway. To assess the relationship between grp and the DNA repair enzymes spn-B or spn-D, the phenotypes produced by the double mutant flies were examined. Neither the dorsal-ventral patterning defects nor the oocyte nuclear morphology defect caused by mutations in the spindle-class genes were suppressed by grp. It is therefore concluded that grp is not the transducer protein in the Drosophila meiotic checkpoint. Similarly, in budding yeast, the checkpoint activity of CHK1 is not required to arrest meiotic cells at pachytene (Abdu, 2002).
Another downstream target of ATM/ATR is Chk2. Chk2 mutants are viable but show defects in maintaining genome stability and are highly sensitive to ionizing radiation. Mutating Chk2 completely blocks DNA damage-induced apoptosis and partially blocks DNA damage-induced cell cycle arrest (Xu, 2001). In budding yeast, the Chk2 homolog, RAD53, is not part of the meiotic checkpoint. However, activation of the pachytene checkpoint is dependent on the activity of Mek1p, a protein highly homologous to Rad53p, both in the kinase domain and in the fork head-associated domain. Thus, it has been suggested that Mek1p functions in the pachytene checkpoint pathway as a counterpart to Rad53p in the DNA damage checkpoint pathway. In fission yeast (Shimada, 2002), the Chk2 homolog, Cds1, and Mek1 are required in the meiotic recombination checkpoint (Abdu, 2002).
To test whether the production of patterning defects by mutations in spindle-class genes is dependent on Chk2, double mutant flies were generated for spn-B, spn-D or okra, and Chk2. A null mutation in Chk2 completely suppressed the dorsal-ventral patterning defects. In double mutant flies, a dramatic increase is observed in the accumulation of Grk protein compared to spn-B single mutants, and an increase is observed in the restoration of dorsal-ventral patterning in the eggshell. A 100% suppression of oocyte nuclear defects is also observed. In wild-type egg chambers, the oocyte nuclear membrane is round and the DNA is located in the center of the nucleus. In contrast, in spn-B mutant egg chambers, the oocyte nuclear DNA is found in a variety of conformations, including wild-type shape (17%), oblong shape (34%), or smaller distinct clumps (49%). In about 90% of the egg chambers, the DNA seems to be attached to the oocyte nuclear membrane. In addition, the morphology of the oocyte nuclear membrane in the mutant egg chambers is also found in a variety of shapes, including round (59%), distorted (32%), or strongly distorted (9%). In egg chambers from the spn-B and Chk2 double mutant flies, the oocyte nuclear membrane and the appearance of the DNA within the oocyte are restored to wild-type (Abdu, 2002).
It was further observed that the posttranslational regulation of Vasa by mei-41 is also dependent on Chk2. Western blot analysis has demonstrated that the altered Vasa protein mobility seen in spn-B ovarian lysates is restored to wild-type in the double mutant flies for spn-B and Chk2. It is concluded that, in Drosophila, activation of Chk2 in the meiotic checkpoint regulates the translation of grk mRNA, apparently through the translational regulator Vasa. However, at this point, it is still not clear how Chk2 may affect translation via Vasa. It was recently reported that Chk2 physically interacts with the Drosophila RNA binding protein Orb (Iwai, 2002). Orb plays roles in localization and translation of several maternal mRNAs, including gurken mRNA. It is therefore possible that Chk2 can directly interact with the translation machinery and can lead to modification of Vasa (Abdu, 2002).
Chk2 is phosphorylated and activated in response to DNA damage by ionizing radiation, UV radiation, and replication block by hydroxyurea. To examine how Chk2 is activated during the meiotic checkpoint, Chk2 expression was examined in spindle-class mutants. Examination of the Chk2 protein by Western blot analysis using an anti-Chk2 antibody revealed that Chk2 appears to be phosphorylated in spn-B mutant ovaries, as evidenced by a more slowly migrating band that can be reduced by alkaline phosphatase treatment. Similar results were also observed when spn-D and okra ovarian extracts were used. The posttranslational modification of Chk2 in spn-B ovarian extracts appears to depend on mei-41, as demonstrated by the shift of Chk2 protein in spn-B and mei-41 double mutant flies back to wild-type-like mobility (Abdu, 2002).
In C. elegans, Chk2 has a role in the initial establishment of pairing between homologous chromosomes and nuclear reorganization during meiotic prophase (Oishi, 2001; MacQueen, 2001). Mutations in Chk2 lead to a lack of chiasmata, inhibition of meiotic crossing over, and nondisjunction. In Drosophila oocytes, meiosis arrests during metaphase I; in crossover-defective and initiation of recombination mutant flies, precocious anaphase is detected. Examination of Chk2 mutant egg chambers shows a wild-type-like metaphase I arrest. No increase was found in X chromosome nondisjunction in Chk2 mutants compared to wild-type. These results suggest that the function of DmChk2 during early meiotic prophase may differ from that of Chk2 in C. elegans (Abdu, 2002).
In the budding yeast, checkpoint-dependent cell cycle arrest at pachytene is achieved by the accumulation of hyperphosphorylated Swe1p, a Wee1-like protein, and subsequent inactivation of Cdc28p. Like other metazoans, Drosophila has two Wee1-like kinases, Wee and Dmyt1. To study the role of Wee in the meiotic checkpoint, the Wee expression in ovaries from spindle-class mutants was compared to expression in wild-type by using an anti-Wee antibody. Western blot analysis shows that the mobility of Wee1 protein is retarded in spn-B, okr, and spn-D mutant ovaries. Wee1 protein also migrates slowly in ovarian extracts prepared from flies mutant for spn-B and grp. In contrast, the mobility of Wee in flies mutant for spn-B and Chk2 is restored to wild-type. Immunohistochemical assays also show an abnormal Wee subcellular localization in spindle-class genes. In wild-type ovaries, Wee protein accumulates inside the oocyte nucleus but is excluded from the DNA, whereas, in about 37% of mutant egg chambers from spn-B, okra, and spn-D, Wee protein accumulates throughout the oocyte nucleus. Interestingly, it was found that mutations in Wee are not able to suppress the dorsal-ventral patterning or the oocyte nuclear morphology defects caused by mutations in the spindle-class genes. Expression of an active form of Cdc2 alone or together with Cyclin A in spn-B mutant flies does not suppress these defects (Abdu, 2002).
The changes in the Wee expression in spindle-class mutants suggest that the initiation of the meiotic checkpoint affects the meiotic cell cycle progression in a Wee-dependent manner, as it does in yeast. However, mutations in Wee1 do not suppress the patterning defects in spindle-class mutants. It is possible that two different pathways are activated by the persistence of unrepaired double-strand DNA breaks, one affecting Wee and the cell cycle, and a second pathway leading to Vasa modification and patterning defects. Alternatively, it is possible that other cell cycle regulators act in parallel to Wee and that the primary effect of the checkpoint is cell cycle arrest, which in turn affects Vasa modification and patterning. However, several studies suggest that, in spindle mutants, there is only a transient cell cycle arrest during early oogenesis, whereas the major effect on translation of grk mRNA occurs during mid-oogenesis. Thus, it is proposed that the patterning defects in spindle mutants are not the result of checkpoint-induced cell cycle arrest (Abdu, 2002).
The p53 tumor suppressor plays a critical role in maintaining genomic stability by regulating cell cycle progression and apoptosis in response to DNA damage. In mice, activation of the meiotic checkpoint by unrepaired DSBs leads to p53-dependent apoptosis. In contrast, the synapsis checkpoint in mice (thought to be a modification of the meiotic checkpoint) eliminates spermatocytes via p53-independent apoptosis. In mammals, p53 is a direct target of Chk2. In flies, p53 is required for the apoptotic response to DNA damage; it undergoes Chk2-dependent modification after DNA damage, and is expressed in the oocyte. In C. elegans, cep-1, a p53 homolog, promotes DNA damage-induced apoptosis and is also required for normal meiotic chromosome segregation in the germline. To assess the role of p53 in the meiotic checkpoint, double mutant flies for the DNA repair enzyme okra (DmRad54) and p53 were generated. The results show that neither the dorsal-ventral patterning defects nor the oocyte nuclear morphology defect caused by mutations in the spindle-class genes are suppressed by p53. From this data, it is concluded that the patterning defects found in the spindle-class mutants are not due to p53 activity (Abdu, 2002).
In summary, the results demonstrate that the Drosophila Chk2 homolog is a transducer of the meiotic checkpoint that is activated by unrepaired double-strand DNA breaks. Activation of Chk2 results in modification of two proteins, Vasa and Wee, which then affect progression of the meiotic cell cycle and translation of gurken mRNA. Wee is, however, not required for the patterning defects seen in the spindle mutations. Activation of the Chk2-dependent meiotic checkpoint may therefore control several cell cycle regulators which in turn may affect both meiosis and translation of gurken mRNA. In particular, it is likely that Wee1 activation regulates cell cycle progression, whereas Chk2 may utilize an independent target to regulate Vasa, which subsequently affects dorsal-ventral patterning as well as nuclear morphology of the oocyte. While dorsal-ventral signaling by Gurken is not a conserved feature of oogenesis found in other organisms, the fact that homologs of Drosophila Chk2 act during meiosis in other organisms raises the possibility that meiotic checkpoints in other species might also act through Chk2 to regulate translation during oogenesis and thus directly link the meiotic cell cycle to the development of the oocyte (Abdu, 2002).
Under normal laboratory conditions chk2null mutant flies produced viable and fertile progeny. Even though the chk2null stock does not grow as well as the Oregon-R stock, this still indicates that chk2 is a nonessential gene that is also not essential for fertility. Is chk2, like its yeast and C. elegans homologs, required for normal recombination and chromosome segregation? In crosses that allowed the scoring of exceptional females and males produced by X chromosome nondisjunction in the F1 progeny, no increase was seen in the rate of X chromosome nondisjunction between chk2null mutants (0/2504) and chk2-/+ heterozygous flies (0/1614). Furthermore, females lacking chk2 displayed no defect in meiotic recombination frequency. In contrast to C. elegans, these results mean that chk2 is not an essential element of the meiotic checkpoint in Drosophila (Masrouha, 2003).
In addition, pattern formation has been linked to the activation of a mei-41-dependent meiotic checkpoint pathway. This pathway is activated in response to unrepaired DSBs during meiosis as they accumulate in several spindle mutants (see Drosophila Okra). To check if chk2 is part of this checkpoint, chk2; spn double mutants were generated and tests were made to determine whether the dorso-ventral patterning defects found in spnB and spnC mutant eggs could be rescued by the removal of chk2. Eggs laid by chk2; spnB and chk2; spnC094 double-mutant females were classified as wild type, fused, or missing dorsal appendages. spn mutant sisters that have one wild-type copy of chk2 were used as internal controls. Furthermore, mei-41; spn and grp; spn double mutants were also generated. Removal of mei-41 rescues the ventralized egg shell phenotype of spn mutants; however, removal of neither chk2 nor grp (chk1) rescues the phenotype. It thus appears that this mei-41-dependent meiotic checkpoint signal is transduced by either a redundant pathway or a chk2-independent mechanism (Masrouha, 2003).
The fact that Chk2 polypeptide expression is very low in the ovary despite the high mRNA levels (Oishi, 1998) may suggest that chk2 mRNA is translationally repressed during oogenesis. UV crosslinking experiments have shown that a polypeptide with the mobility of Orb binds to the chk2 3' untranslated region. To test whether orb is responsible for the low levels of ovarian Chk2 polypeptide, trans-heterozygous females of different combinations of the orb alleles orbF343, orbF303, and orbmel were analyzed for their ovarian Chk2 levels. orbF343 is a strong loss-of-function allele; orbF303 is slightly less severe than orbF343, and orbmel is a weak allele. Quantitative analysis of the Western blot shows that Chk2 polypeptide levels are almost 10-fold higher in orb mutant ovaries than in wild-type ones (Masrouha, 2003).
One of the orb phenotypes is a defect in dorso-ventral patterning of the egg shell. This phenotype can also be caused by the activation of a meiotic checkpoint triggered by unrepaired DNA double-strand breaks. To test whether orb mutations cause a dorso-ventral defect through the over-accumulation of the checkpoint kinase Chk2, chk2; orb double-mutant flies were generated. Double-mutant females were mated to wild-type males and the laid eggs were classified as nonfused, fused, or missing dorsal appendages. As internal controls, orb mutant sisters from the same cross were used; these females had one wild-type copy of chk2. Removing chk2 does not clearly rescue the ventralized egg shell phenotype of orb, indicating that orb mutations cause this defect through another pathway. It thus seems that high levels of Chk2 alone do not induce its cellular function. This is in fact consistent with results from Chk2 orthologs, which show that these kinases need to be phosphorylated (Bartek, 2001b) to be activated (Masrouha, 2003).
To test whether chk2 functions during larval stages in either of these checkpoints, the sensitivity was analyzed of chk2null larvae to MMS, a DNA damage reagent, and to HU, a DNA replication blocking reagent. In this assay, grp mutant flies were used as a positive control, and chk2null and grpfs(A)4 homozygous larvae were generated independently and raised on food supplied with low levels of either HU (20 mM) or MMS (0.08%). Sensitivity is indicated by a preferential loss of homozygous offspring in the presence of MMS and HU. By crossing balanced heterozygous females to balanced heterozygous males, homozygous mutants:heterozygous mutants:Balancer/Balancer offspring are produced at the ratio 1:2:1. Because Balancer/Balancer flies die before they can be scored, a ratio of 1:2 for homozygous:heterozygous mutants would indicate that the mutant flies are fully resistant to the treatment. grp mutants are sensitive to MMS. However, no loss of chk2null mutant flies was observed after treatment with either MMS, indicating that chk2 plays no essential function in these checkpoint pathways during larval development. In contrast to these results, chk2 has been found (Xu, 2001; Peters, 2002) to function in inducing cell cycle arrest and p53-mediated apoptosis upon the activation of a DNA double-strand-break checkpoint in the larval imaginal discs (Masrouha, 2003).
Oishi (1998) reported that both chk2 transcripts and polypeptides are transiently present throughout the embryo at the early syncytial stages, but become restricted to the posterior pole and then to the pole cells (primordial germ cells) by the blastoderm stage where they perdure during gastrulation. On the basis of the expression data above, they proposed that chk2 might play a role in germline establishment and/or maintenance. A chk2null stock was made that can be maintained over generations, indicating that chk2 has no essential role in germline establishment. Pole cells cease dividing after the formation of the cellular blastoderm. They arrest in the G2 phase of the cell cycle until they coalesce with the somatic gonadal precursor cells to form the primitive gonad. To assess the potential involvement of chk2 in greater detail, the number of migrating pole cells in embryos from chk2null mothers was compared to wild type and an average of 24.5 pole cells/embryo was found in the chk2 mutant compared to 37.3 in wild-type. It therefore seems that chk2 plays a role in the maintenance of the germline (Masrouha, 2003).
Genetic and microarray analysis have been used to determine how ionizing radiation (IR) induces p53-dependent transcription and apoptosis in Drosophila. IR induces MNK/Chk2-dependent phosphorylation of p53 without changing p53 protein levels, indicating that p53 activity can be regulated without an Mdm2-like activity. In a genome-wide analysis of IR-induced transcription in wild-type and mutant embryos, all IR-induced increases in transcript levels required both p53 and the Drosophila Chk2 homolog MNK. Proapoptotic targets of p53 include hid, reaper, sickle, and the tumor necrosis factor family member Eiger. Overexpression of Eiger is sufficient to induce apoptosis, but mutations in Eiger do not block IR-induced apoptosis. Animals heterozygous for deletions that span the reaper, sickle, and hid genes exhibited reduced IR-dependent apoptosis, indicating that this gene complex is haploinsufficient for induction of apoptosis. Among the genes in this region, hid plays a central, dosage-sensitive role in IR-induced apoptosis. p53 and MNK/Chk2 also regulate DNA repair genes, including two components of the nonhomologous end-joining repair pathway, Ku70 and Ku80. These results indicate that MNK/Chk2-dependent modification of Drosophila p53 activates a global transcriptional response to DNA damage that induces error-prone DNA repair as well as intrinsic and extrinsic apoptosis pathways (Brodsky, 2004).
Previous studies have established that Drosophila p53 mediates X-irradiation-induced apoptosis and expression of rpr and skl. This study characterized the pathway that transduces the DNA damage signal to the apoptosis and cell cycle machineries. The results indicated that a number of genes in this pathway are largely specific to the cell cycle or apoptotic response. Both cellular assays and transcriptional profiling suggest that Drosophila p53 is required for IR-induced regulation of apoptosis but is not required for G2 arrest. In contrast, mei-41, mus304, and grps were required for cell cycle arrest, but not induction of apoptosis. The biochemical experiments suggested that mnk, which encodes a conserved damage-activated kinase, is required for phosphorylation of p53 following IR. All IR-induced transcription required both mnk and p53. The absence of genes that required mnk or p53 only was consistent with a linear signaling pathway of MNK activating p53, which acts as a global regulator of IR-induced transcription (Brodsky, 2004).
Although mnk and p53 mutant animals have similar defects in IR-induced transcription, mnk also acts in p53-independent pathways. In animals with mutations in double-strand break repair enzymes, unrepaired breaks formed during meiotic recombination activate an mnk-dependent checkpoint signal that disrupts oocyte patterning and nuclear morphology. Induction of the meiotic checkpoint differs from IR-induced transcription in at least two respects: (1) activation of mnk during meiosis requires mei-41, the Drosophila homolog of ATR; (2) p53 is not required for this damage response pathway. In a different damage response pathway, mnk, but not p53, is required for damage-induced inactivation of centrosomes. In this study, IR was found to induced a p53-independent decrease in RNA levels of at least 17 genes, including many developmental regulators. Although this observation could indicate a transcriptional repressor that is regulated by mnk, a model is favored in which an mnk-dependent cell cycle delay following IR has a secondary effect on the developmental induction of these genes. Together, these results and previous studies indicate that mnk regulates multiple signaling pathways in addition to p53-dependent induction of gene expression (Brodsky, 2004).
In mammals, Chk2 and other checkpoint kinases block Mdm2-mediated turnover and inhibition of p53. Several lines of evidence suggest that this regulatory mechanism is not conserved in Drosophila. (1) Simple sequence searches have not revealed an obvious Mdm2 homolog in the Drosophila genome. (2) The Drosophila p53 protein sequence does not contain a conserved binding site for Mdm2. (3) p53 protein levels were not dramatically altered following IR. p53 did exhibit an IR-induced change in gel mobility due to mnk-dependent phosphorylation. Thus, these results provide a clear example of damage-induced activation of p53 without changes in p53 protein levels (Brodsky, 2004).
Phosphorylation of p53 by Chk2 may represent an important step in the evolution of DNA damage responses in multicellular animals. Checkpoint pathways regulating cell cycle control and DNA repair have been highly conserved in eukaryotes, including unicellular organisms such as yeast. In contrast, induction of apoptosis during development or in response to cellular stress is confined to multicellular organisms. p53 phosphorylation by Chk2/MNK was found to be a conserved molecular link between DNA damage detection and the core apoptotic machinery in metazoans. Mdm2 adds an additional layer of complexity to the regulation of mammalian p53 compared to Drosophila p53. Regulation of p53 turnover by Mdm2 may provide mammalian cells with greater control of the levels or timing of p53-dependent transcription (Brodsky, 2004).
Microarray analysis was used to perform a comprehensive analysis of p53 targets following exposure to IR. The number of genes identified in these experiments was substantially smaller than the number of p53 targets identified in mammals. In part, this observation may reflect underlying differences in the damage response pathway in flies and mammals. For example, induction of p21 by mammalian p53 mediates G1 arrest following damage. IR-induced G1 arrest has not been described in Drosophila, consistent with the observation that the Drosophila p21/p27 homolog dacapo is not induced by IR. However, the smaller number of targets identified in Drosophila also reflects experimental differences. Expression changes induced by IR were examined during a defined window of embryonic development. In contrast, targets of mammalian p53 have been identified in many different cell types following different types of DNA damage or simply overexpression of p53. It is likely that additional targets of Drosophila p53 will be identified using other types of cellular stresses in different cell types or developmental stages. For example, UV irradiation of Drosophila embryos has been shown to induce Apaf1 through either E2F or mei-41, depending on the developmental stage (Brodsky, 2004).
The most prominent group of p53 targets identified in this study regulates two apoptotic pathways that are also targeted by mammalian p53. hid, rpr, and skl are part of a group of genes that induce apoptosis by blocking the caspase-inhibiting activity of IAP proteins. Recent experiments have confirmed that HTRA2, a functional homolog of these genes, is a target of mammalian p53. The Drosophila p53 target Eiger is a member of the TNF ligand family and can induce apoptosis when overexpressed. In mammals, FAS and DR5/Killer are p53 targets that can regulate apoptosis by acting as receptors for TNF ligand family members. Thus, two examples of mammalian and Drosophila p53 regulating common signaling pathways have been identified. Combined with the many other proapoptotic targets of mammalian p53, these results support the general hypothesis that multiple components of proapoptotic signaling pathways can be targets for transcriptional regulation following stresses such as DNA damage (Brodsky, 2004).
Although FAS and DR5/Killer are targets of mammalian p53 and act in the extrinsic apoptosis pathway, it is unclear what role they play in DNA damage-induced apoptosis. Analysis of deletion mutations in the Drosophila p53 target Eiger indicates that this gene is not required to initiate IR-induced apoptosis. This negative result is not due to redundancy with a related molecule, since Eiger is the only TNF-related gene in the Drosophila genome sequence. It is possible that the conserved activation of the TNF pathway by p53 is required for the induction of apoptosis under specific conditions not tested in these experiments. Alternatively, induction of Eiger may activate other cellular responses to DNA damage. Further characterization of Eiger function should reveal how cell-cell signaling contributes to survival or genomic stability following DNA damage in multicellular organisms (Brodsky, 2004).
Analysis of the remaining proapoptotic targets of p53 indicates that they are part of a dosage-sensitive mechanism that regulates IR-induced apoptosis. In contrast to Eiger, the proapoptotic genes in the genetic region containing hid, rpr, and skl are both sufficient and necessary for apoptosis. Animals with deletions that include genes in this region are defective in IR-induced apoptosis. Because these proapoptotic genes act, at least in part, by inhibiting a common target (IAP1/Thread), it has been proposed that they contribute to a rheostat-like mechanism in which the added activity of all proapoptotic proteins present must pass a threshold before a cell undergoes the irreversible decision to undergo programmed cell death. Following the observation that three of these genes are induced following DNA damage, the effect of lowering the dose of all proapoptotic genes in this region by half was tested. It was found that deletions in this region were haploinsufficient for IR-induced apoptosis. Dose sensitivity may represent an important feature of damage-induced apoptosis. Animals heterozygous for these deletions exhibit apparently normal morphology and fertility, suggesting that they are not haploinsufficient for developmentally regulated apoptosis. One possible interpretation of these results is that the apoptotic signal in many developmental contexts is well past the threshold required to commit to apoptosis, while the apoptotic signal following DNA damage is closer to that threshold. A lower apoptotic signal following DNA damage may allow cells to monitor DNA repair and block apoptosis if repair is successful. Haploinsufficiency of some tumor suppressor genes, including p53, has been proposed to contribute to cancer development. If stress-induced apoptosis in mammals is sensitive to the dose of p53 target genes, haploinsufficiency of these genes may also contribute to suppression of apoptosis, particularly in cells with extensive aneuploidy (Brodsky, 2004).
Analysis of animals heterozygous for deletions that removed a subset of genes has revealed that loss of one copy of hid is sufficient to reduce IR-induced apoptosis. A greater reduction was observed in larger deletions, indicating that additional genes in this region, likely rpr and skl, also contribute to IR-induced apoptosis. Previous analysis of animals heterozygous for two overlapping deletions [Df(3L)H99 and Df(3L)xr38] that remove both copies of rpr demonstrate reduced levels of IR-induced apoptosis. The current results indicate that part of that reduction is due to haploinsufficiency of hid and other genes in this region. Although the induction of hid RNA was lower than that observed for rpr and skl, hid may exhibit a greater absolute difference in RNA and protein levels following IR. Because null mutations in hid are embryonic lethal, the effects of completely removing hid function were not investigated. The dose-sensitive effects of hid suggest that total loss of hid would completely block IR-induced apoptosis. However, even in animals with normal levels of hid, increased levels of rpr and skl may be required to pass the proapoptotic signaling threshold required for a full DNA damage response. The Ras pathway and a micro-RNA in the bantam locus regulate hid expression. These and other pathways regulating hid may help determine which cells in the developing wing are most sensitive to DNA damage (Brodsky, 2004).
The other class of p53 targets identified in these experiments includes components of the Ku and Mre11 DNA repair complexes. Both of these complexes participate in repair of double-strand DNA breaks by nonhomologous end joining (NHEJ). Compared with homologous recombination, NHEJ is a potentially error-prone mechanism for DNA repair. Mutagenic DNA repair mechanisms are a prominent feature of the SOS response in bacteria that apparently promotes cell survival following DNA damage at the expense of genomic integrity. The ability of multicellular animals to eliminate damaged cells by apoptosis might suggest that low-fidelity mechanisms of DNA repair would not be favored following damage. However, the induction of NHEJ components by p53 suggests that mechanisms such as apoptosis or cell cycle arrest that are presumed to prevent mutations following DNA damage may compete with mechanisms that promote cell survival and prevent aneuploidy by error-prone DNA repair. The previous demonstration that an isoform of Ku86 is also a target of mammalian p53 suggests that this is an evolutionarily conserved response to DNA damage in metazoans that may modulate mutagenesis following DNA damage (Brodsky, 2004).
Ionizing radiation (IR) can induce apoptosis via p53, which is the most commonly mutated gene in human cancers. Loss of p53, however, can render cancer cells refractory to therapeutic effects of IR. Alternate p53-independent pathways exist but are not as well understood as p53-dependent apoptosis. Studies of how IR induces p53-independent cell death could benefit from the existence of a genetically tractable model. In Drosophila, IR induces apoptosis in the imaginal discs of larvae, typically assayed at 4-6 hr after exposure to a LD50 dose. In mutants of Drosophila Chk2 or p53 homologs, apoptosis is severely diminished in these assays, leading to the widely held belief that IR-induced apoptosis depends on these genes. This study shows that IR-induced apoptosis still occurs in the imaginal discs of chk2 and p53 mutant larvae, albeit with a delay. This phenomenon is a true apoptotic response because it requires caspase activity and the chromosomal locus that encodes the pro-apoptotic genes reaper, hid, and grim. Chk2- and p53-independent apoptosis is IR dose-dependent and is therefore probably triggered by a DNA damage signal. It is concluded that Drosophila has Chk2- and p53-independent pathways to activate caspases and induce apoptosis in response to IR. This work establishes Drosophila as a model for p53-independent apoptosis, which is of potential therapeutic importance for inducing cell death in p53-deficient cancer cells (Wichmann, 2006).
The Drosophila homologs of Chk2 and p53 are required, not for induction of apoptosis, but for timely induction of apoptosis in response to irradiation. Radiation-induced cell death still occurs in chk2 and p53 mutants, albeit with a delay. Four lines of evidence support the idea that this delayed cell death is apoptosis rather than necrosis: (1) it is detected by staining with AO, which has been shown to stain apoptotic but not necrotic cells; (2) it accompanies activation of caspases, a hallmark of apoptosis but not necrosis; (3) it requires caspase activity, which is required for apoptosis but not necrosis, and (4) it requires the chromosomal locus encoding the proapoptosis genes rpr, hid, and grim, whose protein products are required to inhibit DIAP1 and activate caspases. These results indicate that there is a Chk2-/p53-independent pathway that commits damaged cells to apoptosis and utilizes many of the same downstream components as the Chk2-/p53-dependent apoptosis pathway (Wichmann, 2006).
Two lines of evidence support the idea that DNA damage is the signal that induces Chk2-/p53-independent apoptosis after exposure to ionizing radiation. First, the amount of Chk2-/p53-independent apoptosis appears to increase with IR dose. This dose dependence suggests that the amount of DNA damage is what induces Chk2-/p53-independent apoptosis but does not rule out the contribution of other damages that result from IR. Second, higher levels of Chk2-/p53-independent apoptosis are observed when the ability to repair DNA is compromised, as in mei-41, p53 double mutants. Collectively, these data suggest that DNA damage caused by x-rays induces Chk2-/p53-independent apoptosis (Wichmann, 2006).
IR-induced apoptosis in chk2 and p53 mutants shows a temporal delay. IR-induced apoptosis is also delayed in H99 heterozygotes, possibly because H99 heterozygotes contain half the gene dose of the proapoptotic Smac/Diablo orthologs and it may take longer for the proapoptotic gene products to accumulate to the point of an apoptosis-stimulating threshold. IR induced increase in the transcripts of rpr and hid, two of the H99-encoded genes, still occurred in chk2 (rpr and hid) and p53 (hid) mutants, but to lower levels (for rpr) and after a delay. Therefore, apoptosis may be delayed in chk2 and p53 mutants because proapoptotic gene products take longer to accumulate to a threshold in the absence of Chk2 or p53 regulation. The data showing that IR-induced apoptosis is further delayed in a chk2, H99/+ double mutant, compared with a chk2 single mutant, support this claim. Furthermore, the results suggest the existence of at least another signaling pathway that does not operate through Chk2 or p53, but nonetheless links the same signal (DNA damage) to a similar outcome (accumulation of H99-encoded gene products) (Wichmann, 2006).
RT-PCR experiments revealed interesting differences in the identity and onset of induction of proapoptotic genes in chk2 and p53 mutants. rpr and hid are induced at 4 hr after irradiation in chk2 mutants, whereas hid and skl are induced between 12 and 18 hr after irradiation in p53 mutants. The basis for these differences is not understood. More detailed time courses as well as deletion analysis of the H99 locus to determine the contribution of each proapoptotic gene to Chk2-/p53-independent apoptosis needs to be performed to address these issues (Wichmann, 2006).
The data presented in this study establish Drosophila as a model for studying p53-independent apoptosis. p53 is the most commonly mutated gene in human cancers. Loss of p53 poses an immense clinical problem because p53-deficient cancer cells no longer stimulate p53-dependent apoptosis in response to radiation or genotoxic chemotherapy drugs. In this scenario, p53-independent apoptotic pathways become key for inducing cancerous cells to die because they provide potential therapeutic targets. In mammals, a p53-independent apoptosis pathway that is mediated by p73, another member of the p53 family, has been identified. In Drosophila, Dmp53 is the only known p53 family member. Therefore, the p53-independent apoptosis that was identified and characterized in this article is likely to represent a previously unknown process. An important goal in the future will be to dissect the Chk2-/p53-independent pathway that links DNA damage to the proapoptotic genes of the H99 locus (Wichmann, 2006).
Several candidates were tested and eliminated as regulators of Chk2-/p53-independent cell death. Mei-41 (ATR) is not required for Chk2-/p53-independent cell death because mei-41, p53 double mutants actually exhibit more cell death than p53 alone. Recent work showed that ectopic induction of eiger, a TNF ligand homolog, can induce apoptosis in Drosophila. Chk2-/p53-independent cell death still occurs in p53, eiger double mutants, suggesting that the TNF pathway is not involved in the induction of cell death characterized in this study. Work in mammalian cells showed that overexpression of c-Myc can induce p53-independent apoptosis. Chk2-/p53-independent apoptosis still occurs in Dmyc, p53 double mutants, indicating that Dmyc is not required for this response (Wichmann, 2006).
A classical genetic screen may identify components of the Chk2-/p53-independent apoptosis pathway, as well as testing more candidates, such as the transcription factor de2f1, grapes (DmChk1), DmATM, and genes required for autophagy. Autophagic cell death, in which a cell lyses itself, occurs during Drosophila metamorphosis to lyse polyploid tissues such as the salivary glands and the fat body and provide nutrients for diploid cells of the imaginal discs; autophagy has been described in larvae only in the polyploid cells and only in response to starvation. Nonetheless, autophagy shares characteristics with apoptosis, including being detectable by AO staining and being dependent on caspases and the H99 locus, and for this reason remains a formal possibility (Wichmann, 2006).
In conclusion, studies have shown that IR-induced apoptosis in two key models for apoptosis, C. elegans and Drosophila, depends on p53. This study has provided evidence that, contrary to the accepted view, Chk2 and p53 are not required for radiation-induced cell death in Drosophila. Furthermore, normal timing of apoptosis that depends on Chk2 and p53 is also not required for ensuring survival after irradiation. Radiation-induced cell death that is independent of Chk2 and p53 depends on radiation dose, has characteristics of apoptosis and is likely to rely on a novel mechanism(s) because no other members of the p53-family are known in Drosophila. This work is the first to establish Drosophila as a model for p53-independent apoptosis. Identification of genes required for Chk2-/p53-independent cell death in Drosophila is of potential therapeutic value because protein products of their human homologs may represent novel targets that can be activated clinically to eliminate p53-deficient cancer cells (Wichmann, 2006).
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).
Gametogenesis is a highly regulated process in all organisms. In Drosophila, a meiotic checkpoint which monitors double-stranded DNA breaks and involves Drosophila ATR and Chk2 coordinates the meiotic cell cycle with signaling events that establish the axis of the egg and embryo. Checkpoint activity regulates translation of the transforming growth-factor-alpha-like Gurken signaling molecule which induces dorsal cell fates in the follicle cells. Mutations in the Drosophila gene cutoff (cuff) affect germline cyst development and result in ventralized eggs as a result of reduced Grk protein expression. Surprisingly, cuff mutations lead to a marked increase in the transcript levels of two retrotransposable elements, Het-A and Tart. Small interfering RNAs against the roo element are still produced in cuff mutant ovaries. These results indicate that Cuff is involved in the rasiRNA pathway and most likely acts downstream of siRNA biogenesis. The eggshell and egg-laying defects of cuff mutants are suppressed by a mutation in chk2. Mutations in aubergine (aub), another gene implicated in the rasiRNA pathway, are significantly suppressed by chk2 mutation. These results indicate that mutants in rasiRNA pathways lead to elevated transposition incidents in the germline, and that this elevation activates a checkpoint that causes a loss of germ cells and a reduction of Gurken protein in the remaining egg chambers (Chen, 2007).
cutoff (cuff) mutations were isolated in a large-scale female-sterile screen of Drosophila, and one additional allele was identified in a screen for P element insertions. Females transheterozygous for cuff alleles lay eggs with various degrees of ventralization. The dorsoventral polarity of the egg and embryo depends on the levels of the Gurken (Grk) ligand, which is produced and secreted by the germline and activates the EGF receptor (Egfr) in the overlying follicle cells. To determine whether Grk-Egfr signaling was affected, the grk expression pattern was analyzed in a strong cuff mutant background. In wild-type egg chambers at stage 9 of oogenesis, grk RNA becomes restricted to the future dorsal-anterior side of the oocyte and forms a cap around the oocyte nucleus. Grk protein is translated from the tightly localized RNA and is also spatially restricted to the membrane overlying the oocyte nucleus. cuff mutants do not significantly disrupt grk RNA localization. However, in many mid-stage egg chambers, the Grk protein level is greatly reduced, such that between 10% and 40% of the egg chambers contain no detectable Gurken protein at all, consistent with defects in grk translation. In wild-type egg chambers by stage 3 of oogenesis, the oocyte nucleus forms a compact structure termed the karyosome. In cuff mutants, karyosome formation is affected in 10%–20% of the egg chambers, in which the DNA assumes various shapes and is often found in separate clumps (Chen, 2007).
Genomic database searches identified the yeast gene Rai1 as a homolog of cuff. This gene has been shown to interact with a nuclear 5′–3′ exoribonuclease (Rat1) that is involved in rRNA processing and transcriptional termination. A cytoplasmic homolog of Rat1, Xrn1, has also been described in yeast and vertebrates and has been implicated in mRNA regulation that is localized to cytoplasmic processing bodies. An HA-tagged Drosophila Rat1 (CG10354) construct was generated and overexpressed with a fully functional FLAG-tagged Cuff in the ovary. Using immunoprecipitation (IP), no any interaction between the exoribonuclease and Cuff was detected. It is therefore possible that Drosophila Rat1 is not the correct partner for Cuff. This is also supported by the observation that overexpressed Rat1, as expected, localizes to the nucleus, whereas overexpressed Cuff localizes to the cytoplasm. It was not possible to to detect endogenous Cuff protein with an anti-Cuff antibody, presumably because of low levels of protein expression. However, overexpressed HA-tagged Cuff partially colocalizes with perinuclear puncta in the nurse cells in younger egg chambers. A similar localization pattern has been described for the helicase Vasa, and it was found that Cuff partially colocalizes with Vasa in the cytoplasm. The perinuclear localization pattern, also designated as nuage in the germ cells and related to mammalian P bodies, has been described for components of the RNAi machinery and for genes involved in RNA degradation (Chen, 2007).
Given the eggshell ventralization and the karyosome defect, cuff has mutant phenotypes similar to those of a group of mutants known as the spindle-class genes. Several members of this group encode DNA-repair genes, for instance, spindle(spn) B (XRCC3) and okra (DmRad54). In these mutants, the DSBs that are created during recombination persist and thus activate Chk2 through the Drosophila ATR homolog mei-41. The activity of these kinases negatively regulates the translation of Grk, possibly through a posttranslational modification of Vasa; this modification in turn leads to ventralization of the eggs laid by mutant females. Inactivation of the checkpoint, for instance through mutations in chk2 or mei-41, suppresses the eggshell defects of the spindle-class DNA-repair mutants. In addition, in double mutants of the DNA-repair genes and the genes required for initiating the DSBs, such as c(3)g, mei-W68, or mei-P22, DSBs are not generated; therefore, the checkpoint is not activated, and the eggshell morphology is normal, even in the presence of the repair mutants. To check whether Cuff is involved in the repair of DSBs initiated in prophase of meiosis I, mei41;cuff and cuff;c(3)g double mutants were generated. Although both mutations suppress the eggshell defect of spnB or okra to wild-type morphology, neither suppresses the eggshell defect of cuff, indicating that Cuff does not function in the meiotic repair pathway. Surprisingly, however, a mutation in chk2 partially suppresses the eggshell defect of cuff as well as the defects in cyst development. chk2 cuff double mutants lay mostly wild-type-looking eggs, and have cysts with highly branched fusomes in the germaria, and the females lay more eggs than cuff single mutants, although the rescue is not 100%. In certain allelic combinations, it was possible to observe a dominant effect in the chk2 suppression of the cuff eggshell defect (Chen, 2007).
Previous work has suggested the DNA-repair checkpoint, upon activation, regulates Grk translation through a posttranslational modification of Vas, and that this modification results in slower Vas electrophoretic mobility. To address whether the checkpoint acts in the same manner in cuff mutants, Vas mobility was assayed in cuff mutant combinations. In cuff mutants, Vas migrates slightly more slowly than wild-type control, consistent with the modification seen in the DNA-repair mutants. The mobility is not changed in mei41;cuff double-mutant background, which is consistent with the fact that mei41 mutants do not significantly suppress the eggshell phenotype of cuff. However, Vasa mobility is restored to wild-type in the chk2 cuff double mutant. This suggests that although the checkpoint is activated through a different sensing mechanism in cuff mutants, upon activation the checkpoint involves Chk2 and acts through similar pathways to affect Gurken translation in the egg chambers that escape the early arrest (Chen, 2007).
Several of the spindle-class genes, such as spnE and aub, have been shown to be essential components of the RNAi machinery. Because overexpressed Cuff has a perinuclear localization, whether Cuff might also be required in RNAi pathways was tested. Recently, a specific branch of the RNAi pathways, that involving the repeat-associated small interfering RNA (rasiRNA), has been implicated in the control of retrotransposable elements in the Drosophila germline. Using qRT-PCR, the level of Het-A and Tart, two of the retrotransposable elements responsible for maintaining the telomere in Drosophila, was studied. Previously, it has been shown that in spnE and aub mutants, Het-A and Tart transcripts are derepressed and that this derepression results in a marked elevation in the transcripts level. Compared with heterozygous controls, spnE homozygous mutant females have Het-A and Tart transcript levels that are upregulated by approximately 10-fold, whereas in aub mutants only Het-A is significantly upregulated. In cuff mutant females, the elevation for both transcripts is even more pronounced. Compared with Het-A levels in the heterozygous control, those in cuff mutants are elevated more than 800-fold, and Tart transcript levels increase by more than 20-fold. Transposable elements are normally silenced in the Drosophila germline by the rasiRNA pathway; this silencing process appears to be strongly impaired in the cuff mutants. Whether the upregulation of the transposable elements in cuff mutants could be due to a reduction in the level of rasiRNAs was further tested. However, it was found that the levels of the 25-nt-long roo interfering RNA are not reduced in cuff mutant ovaries, in contrast to ovaries mutant for aub. This indicates that Cuff is not involved in the biogenesis of the rasiRNAs and points to a function for Cuff in the actual silencing process. Because high transcript levels of the retrotransposable elements in the germline are correlated with elevated transposition incidents, which in turn lead to decreased chromosomal integrity, it is possible that such chromosomal defects activate the checkpoint involving chk2. In addition, because transposable elements are involved in the regulation of chromatin structure, the existence of a chromatin checkpoint that involves Chk2 activity is also possible. Once Chk2 is activated, either by the mutants in DNA-repair pathways or by RNAi components such as Cuff and Aub, Chk2 activity leads to posttranslational Vas modification and a negative regulation of Grk translation. However, unlike DNA repair mutations, cuff and aub mutations are not suppressed to wild-type morphology and fecundity by mutations in mei41, suggesting that they activate the checkpoint through a different, or additional, sensing mechanism. Furthermore, most of the mutants in DNA-repair pathways do not cause defects in cyst development or germline stem cell maintenance. These additional defects seen in cuff mutants could be due to the timing of checkpoint activation. DNA-repair mutants activate the meiotic checkpoint during meiotic prophase, which initiates after the formation of the 16 cell cyst, whereas cuff and aub mutants appear to act earlier in oogenesis, given that they already have effects during the mitotic cycles preceding the onset of meiosis. The transposon-activated checkpoint leads not only to translational arrest of Grk, but also to mitotic cell-cycle arrest. Many of the arrested germline cells and cysts eventually undergo apoptosis, leading to gradual loss of both germline stem cells and developing cysts in cuff mutants. However, germ cells that escape the early arrest encounter the second checkpoint effect, which leads to a reduction in Gurken translation (Chen, 2007).
It was recently discovered that there are a large number of different small RNAs generated in the germline of both mammals and flies. Many of them are associated with Piwi family proteins, and most have no known functions. Because the germline represents a special cell type that will pass its DNA on to future progeny, it is possible that selfish elements have developed a high propensity to remobilize in the germline. Furthermore, it is very plausible that in most organisms the germline has evolved sophisticated mechanisms to defend itself against such transposable elements. Many of the small RNAs found in the germline may be involved in the defense against transposable elements, as well as in the regulation of transcription and translation. When the machinery to generate these small silencing RNAs or the effector complexes that are responsible for transcript degradation are disrupted, chromosomal integrity might be at risk. This study has found that a checkpoint involving the conserved Chk2 kinase monitors the RNAi-mediated events in the Drosophila germline and ensures the genomic integrity of the progeny. Chk2 therefore acts as a surveillance factor for both transposon-generated problems as well as DNA-repair problems in the germline. Whether Chk2 has a similar role in the mammalian germline will be interesting to investigate in the future (Chen, 2007).
The 13 syncytial cleavage divisions that initiate Drosophila embryogenesis are under maternal genetic control. The switch to zygotic regulation of development at the midblastula transition (MBT) follows mitosis 13, when the cleavage divisions terminate, transcription increases and the blastoderm cellularizes. Embryos mutant for grp, which encodes Checkpoint kinase 1 (Chk1), are DNA-replication-checkpoint defective and fail to cellularize, gastrulate or to initiate high-level zygotic transcription at the MBT. The mnk (also known as loki) gene encodes Checkpoint kinase 2 (Chk2), which functions in DNA-damage signal transduction. mnk grp double-mutant embryos are replication-checkpoint defective but cellularize, gastrulate and activate high levels of zygotic gene expression. grp mutant embryos accumulate DNA double-strand breaks and DNA-damaging agents induce a mnk-dependent block to cellularization and zygotic gene expression. It is concluded that the DNA-replication checkpoint maintains genome integrity during the cleavage divisions, and that checkpoint mutations lead to DNA damage that induces a novel Chk2-dependent block at the MBT (Takada, 2007).
Studies in lower eukaryotes initially defined checkpoints as< non-essential pathways that delay cell cycle progression in response to external stress. Subsequently, checkpoint mutations in higher eukaryotes were found to induce developmental defects and embryonic lethality. In Drosophila, the DNA-replication checkpoint is required to delay the cell cycle during the late cleavage stage, and checkpoint mutants subsequently fail to cellularize or activate zygotic gene expression at the MBT. These findings suggested that the replication checkpoint has a direct role in metazoan developmental. Alternatively, the observed developmental defects could be an indirect consequence of checkpoint failure (Takada, 2007).
A null mutation in mnk, which encodes the conserved DNA-damage signaling kinase Chk2, efficiently suppresses the cellularization and zygotic gene-activation defects in grp, but does not restore wild-type cell cycle timing or replication-checkpoint function. It is therefore concluded that progression through the Drosophila MBT does not directly require Chk1 or checkpoint-dependent cell cycle delays. Instead, the data indicate that the essential function for the replication checkpoint is to prevent DNA damage during the syncytial blastoderm divisions, which triggers a Chk2-dependent block to zygotic gene activation and cellularization. Supporting this proposal, DNA-damaging agents trigger a Chk2-dependent block to cellularization and zygotic gene activation, and grp mutations accumulate DNA double-strand breaks. Chk2 is likely to have multiple targets during this developmental response to DNA damage; these targets may include transcription factors that control the expression of genes implicated in cell cycle control and cellularization (Takada, 2007).
Embryos mutant for grp or mei-41 lack a functional replication checkpoint and progress into mitosis prior to S-phase completion, triggering defects in γ-Tubulin localization and microtubule nucleation. These mitotic defects are suppressed by mnk, raising the possibility that mnk suppresses the grp mutant developmental block at the MBT by restoring mitotic function. However, mnk does not suppress the chromosome-segregation defects associated with grp mutants. More significantly, inducing DNA damage following the final syncytial blastoderm division triggers a Chk2-dependent block to cellularization. DNA damage can therefore induce a Chk2-dependent developmental block that is distinct from the damage and Chk2-dependent block to mitosis (Takada, 2007).
The studies outlined here support a simple model in which the developmental arrest associated with grp mutations results from defects in the established function for this kinase in cell cycle control. The early cleavage-stage divisions have a simplified S-phase/M-phase cell cycle, and it is proposed that the crucial function of Chk1 is to delay mitosis until DNA replication is complete. In grp mutants, progression into mitosis before replication is complete leads to DNA damage, which activates a Chk2-dependent block to developmental progression. Intriguingly, disrupting Chk1 function also leads to early embryonic lethality in frogs, mice and worms. Chk1 knockdown in Xenopus and Chk1 (also known as Chek1 - Mouse Genome Informatics) mutations in mouse lead to apoptotic death of the embryo, consistent with a DNA-damage response. It is therefore speculated that Chk1 has a conserved function in maintaining genome integrity during the cleavage stage, and that the early embryonic lethality in checkpoint mutants is a consequence of DNA-damage signaling (Takada, 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).
Mutation of human microcephalin (MCPH1) causes autosomal recessive primary microcephaly, a developmental disorder characterized by reduced brain size. mcph1, the Drosophila homolog of MCPH1, has been identified in a genetic screen for regulators of S-M cycles in the early embryo. Embryos of null mcph1 female flies undergo mitotic arrest with barrel-shaped spindles lacking centrosomes. Mutation of Chk2 suppresses these defects, indicating that they occur secondary to a previously described Chk2-mediated response to mitotic entry with unreplicated or damaged DNA. mcph1 embryos exhibit genomic instability as evidenced by frequent chromatin bridging in anaphase. In contrast to studies of human MCPH1, the ATR/Chk1-mediated DNA checkpoint is intact in Drosophila mcph1 mutants. Components of this checkpoint, however, appear to cooperate with MCPH1 to regulate embryonic cell cycles in a manner independent of Cdk1 phosphorylation. A model is proposed in which MCPH1 coordinates the S-M transition in fly embryos: in the absence of mcph1, premature chromosome condensation results in mitotic entry with unreplicated DNA, genomic instability, and Chk2-mediated mitotic arrest. Finally, brains of mcph1 adult male flies have defects in mushroom body structure, suggesting an evolutionarily conserved role for MCPH1 in brain development (Rickmyre, 2007).
Several studies have implicated human MCPH1 in the cellular response to DNA damage. The DNA checkpoint is engaged at critical cell-cycle transitions in response to DNA damage or incomplete replication and serves as a mechanism to preserve genomic integrity. Triggering of this checkpoint causes cell-cycle delay, presumably to allow time for correction of DNA defects. When a cell senses DNA damage or incomplete replication, a kinase cascade is activated. Activated ATM and ATR kinases phosphorylate their targets, including the checkpoint kinase Chk1, which is activated to phosphorylate its targets. The first clue that MCPH1 plays a role in the DNA damage response came from siRNA-mediated knockdown studies in cultured mammalian cells demonstrating a requirement for MCPH1 in the intra-S phase and G2-M checkpoints in response to ionizing radiation. Two recent reports have further implicated MCPH1 in the DNA checkpoint, although puzzling discrepancies remain to be resolved. One report indicates that MCPH1 functions far downstream in the pathway, at a level between Chk1 and one of its targets, Cdc25. Another report suggests that MCPH1 is a proximal component of the DNA damage response required for radiation-induced foci formation (i.e. recruitment of checkpoint and repair proteins to damaged chromatin) (Rickmyre, 2007).
Additional functions have been reported for MCPH1. MCPH1- lymphocytes of microcephalic patients exhibit premature chromosome condensation (PCC) characterized by an abnormally high percentage of cells in a prophase-like state, suggesting that MCPH1 regulates chromosome condensation and/or cell-cycle timing. A possible explanation for the PCC phenotype is that MCPH1-deficient cells have high Cdk1-cyclin B activity, which drives mitotic entry; decreased inhibitory phosphorylation of Cdk1 was found to be responsible for elevated Cdk1 activity in MCPH1-deficient cells. It is not clear whether MCPH1's role in regulating mitotic entry in unperturbed cells is related to its checkpoint function; intriguingly, Chk1 has similarly been reported to regulate timing of mitosis during normal division. MCPH1 (also called Brit1) was independently identified in a screen for negative regulators of telomerase, suggesting that it may function as a tumor suppressor. Further evidence for such a role comes from a study showing that gene copy number and expression of MCPH1 is reduced in human breast cancer cell lines and epithelial tumors (Rickmyre, 2007).
This study reports the identification and phenotypic characterization of Drosophila mutants null for mcph1. Syncytial embryos from mcph1 females exhibit genomic instability and undergo mitotic arrest due to activation of a DNA checkpoint kinase, Chk2. On contrast to reports of MCPH1 function in human cells, the ATR/Chk1-mediated DNA checkpoint is intact in Drosophila mcph1 mutants. It is propose that Drosophila MCPH1, like its human counterpart, is required for proper coordination of cell-cycle events; in early embryos lacking mcph1, chromosome condensation prior to completion of DNA replication causes genomic instability and Chk2-mediated mitotic arrest (Rickmyre, 2007).
Drosophila mcph1 was identified in a genetic screen for cell-cycle regulators, and it is required for genomic stability in the early embryo. Three additional primary microcephaly (MCPH) genes have been identified in humans: ASPM, CDK5RAP2, and CENPJ. Much understanding of the biological functions of the proteins encoded by human MCPH genes has come from studies of their Drosophila counterparts. Mutation of abnormal spindle (asp), the Drosophila ortholog of ASPM, results in cytokinesis defects and spindles with poorly focused poles. The Drosophila ortholog of CDK5RAP2, centrosomin (cnn), is required for proper localization of other centrosomal components. Sas-4, the Drosophila ortholog of CENPJ, is essential for centriole production, and the mitotic spindle is often misaligned in asymmetrically dividing neuroblasts of Sas-4 larvae. Whereas all of these primary microcephaly genes are critical regulators of spindle and centrosome functions, mitotic defects in Drosophila mcph1 mutants are largely secondary to Chk2 activation in response to DNA defects; thus, mcph1 probably represents a distinct class of primary microcephaly genes (Rickmyre, 2007).
MCPH1 is a BRCT domain-containing protein, suggesting that it plays a role in the DNA damage response. Conflicting models of MCPH1 function, however, have emerged from studies of human cells as it has been proposed to function at various levels in this pathway: upstream, at the level of damage-induced foci formation (Rai, 2006) and further downstream, to augment phosphorylation of targets by the effector Chk1 (Alderton, 2006). The phenotype of embryos from null mcph1 females is more severe than that of embryos from null grp females, suggesting that enhancement of phosphorylation of GRP (Chk1) substrates is not the sole function of MCPH1. Furthermore, both the DNA checkpoint in larval stages and its developmentally regulated use at the MBT are intact in mcph1 mutants, suggesting a requisite role for MCPH1 in the DNA checkpoint evolved in higher organisms (Rickmyre, 2007).
Studies of human cells suggest a role for MCPH1 in regulation of chromosome condensation. Microcephalic patients homozygous for a severely truncating mutation in MCPH1 show increased frequency of G2-like cells displaying premature chromosome condensation (PCC) with an intact nuclear envelope. Depletion of Condensin II subunits by RNAi in MCPH1-deficient cells leads to reduction in the frequency of PCC, suggesting that MCPH1 is a negative regulator of chromosome condensation. A decreased level of inhibitory phosphates on Cdk1 that correlated with PCC have been observed in MCPH1-deficient cells. It has been proposed that MCPH1 maintains Cdk1 phosphorylation in an ATR-independent manner because PCC is not seen in cells of patients with Seckel syndrome, which is caused by mutation of ATR; residual ATR present in these cells, however, may be sufficient to prevent PCC. Furthermore, in several experimental systems, ATR and Chk1 have been implicated in an S-M checkpoint that prevents premature mitotic entry with unreplicated DNA (Rickmyre, 2007).
This study has shown that embryos from grp (Chk1) females occasionally undergo mcph1-like arrest in early syncytial cycles, prior to the time at which inhibitory phosphorylation of Cdk1 is thought to control mitotic entry. Thus, decreased signaling through the DNA checkpoint resulting in less Cdk1 phosphorylation is unlikely to explain this mcph1-like arrest. In contrast to studies of MCPH1-deficient human cells, no decrease is detected in pY15-Cdk1 levels in mcph1 embryos allowed to progress beyond their normal arrest point by mutation of mnk (Chk2). Based on these data and the PCC phenotype associated with loss of MCPH1 in humans, a model is proposed in which MEI-41/GRP cooperate with MCPH1 in syncytial embryos in a Cdk1-independent manner to delay chromosome condensation until DNA replication is complete. In the absence of mcph1, it is hypothesized that embryos condense chromosomes before finishing S phase, resulting in DNA defects (bridging chromatin), Chk2 activation, and mitotic arrest. It was not possible to directly monitor chromosome condensation in mnk mcph1 embryos expressing Histone-GFP, because fly stocks could not be established carrying this transgene in the mnk background. Live imaging of mcph1 embryos was not technically feasible because they arrest prior to cortical stages, and yolk proteins obscure more interior nuclei in early embryos. grp embryos have been reported to initiate chromosome condensation with normal kinetics, although a subtle PCC phenotype might be difficult to detect (Rickmyre, 2007).
Support for a model that MCPH1 allows completion of S phase by delaying chromosome condensation comes from the observation that inhibition of DNA replication in syncytial embryos (via injection of aphidicolin or HU) results in phenotypes similar to those observed in mcph1 embryos, including chromatin bridging, which is presumably a direct consequence of progressing through mitosis with unreplicated chromosomes, and Chk2 activation. Alternatively, mcph1 might be required during S phase for timely completion of DNA synthesis; in this case, mcph1 embryos would initiate chromosome condensation with normal kinetics prior to completing replication. Coordination of S-phase completion and mitotic entry may be particularly critical in the rapid cell cycles of the early embryo that lack gap phases and may explain why loss of Drosophila mcph1 is most apparent at this developmental stage. Interestingly, even in the absence of exogenous genotoxic stress, MCPH1-deficient human cells also exhibit a high frequency of chromosomal aberrations, which may be a consequence of PCC (Rickmyre, 2007).
An evolutionary role for mcph1 in expansion of brain size along primate lineages has emerged in recent years. In brains of Drosophila mcph1 males, low-penetrance defects in MB structure were found. Both MCPH1 isoforms are expressed in larval brains, and all mcph1 mutations described in this study affect both isoforms, so it is unclear whether MB formation requires one or both isoforms. The lack of MB defects in mcph1 females is puzzling because both isoforms are found in male and female larval brains; other sex-specific factors are probably involved. Larval brains of mcph1 males show no obvious aneuploidy or spindle orientation defects, so the cellular basis for these defects remains to be determined. It will be interesting to test in future studies whether mei-41 and grp, which cooperate with mcph1 to regulate early embryogenesis, are similarly required in Drosophila males for brain development (Rickmyre, 2007).
In conclusion, this study has demonstrated an essential role for Drosophila MCPH1 in maintaining genomic integrity in the early embryo. The data suggest that, in contrast to the mammalian protein, Drosophila MCPH1 is not required for the DNA checkpoint, although its role in regulating other processes (e.g. chromosome condensation) may be conserved. It is predicted that the early embryo of Drosophila will continue to be an important model genetic system for unraveling the biological functions of MCPH1, a critical determinant of brain size in humans (Rickmyre, 2007).
In a screen for cell-cycle regulators, a Drosophila maternal effect-lethal mutant was identified named 'no poles' (nopo). Embryos from nopo females undergo mitotic arrest with barrel-shaped, acentrosomal spindles during the rapid S-M cycles of syncytial embryogenesis. CG5140, which encodes a candidate RING domain-containing E3 ubiquitin ligase, was identified as the nopo gene. A conserved residue in the RING domain is altered in the EMS-mutagenized allele of nopo, suggesting that E3 ligase activity is crucial for NOPO function. Mutation of a DNA checkpoint kinase, CHK2, suppresses the spindle and developmental defects of nopo-derived embryos, revealing that activation of a DNA checkpoint operational in early embryos contributes significantly to the nopo phenotype. CHK2-mediated mitotic arrest has been shown to occur in response to mitotic entry with DNA damage or incompletely replicated DNA. Syncytial embryos lacking NOPO exhibit a shorter interphase during cycle 11, suggesting that they may enter mitosis prior to the completion of DNA replication. Bendless (Ben), an E2 ubiquitin-conjugating enzyme, interacts with NOPO in a yeast two-hybrid assay; furthermore, ben-derived embryos arrest with a nopo-like phenotype during syncytial divisions. These data support the model that an E2-E3 ubiquitination complex consisting of Ben-Uev1A (E2 heterodimer) and Nopo (E3 ligase) is required for the preservation of genomic integrity during early embryogenesis (Merkle, 2009).
To ensure faithful transmission of the genome upon cell division, eukaryotic cells have developed checkpoints, regulatory pathways that delay cell-cycle progression until completion of prior events. The DNA damage/replication checkpoint plays a crucial role in preserving genomic integrity. Upon detection of DNA defects, the kinases ATM (ataxia telangiectasia mutated) and ATR (ATM-Rad3-related) are recruited to sites of damage and activated. ATM and ATR substrates include checkpoint kinases CHK1 and CHK2, which phosphorylate proteins that mediate cell-cycle arrest. The ensuing delay, resulting from engagement of this checkpoint, presumably allows cells time to correct defects (Merkle, 2009).
Research over the past decade has highlighted major roles for protein ubiquitination in regulating cellular responses to DNA damage. This post-translational modification, which involves covalent linkage of one or more ubiquitin molecules to another protein, regulates many fundamental cellular processes. Ubiquitination may alter the fate of a protein in numerous ways, such as targeting it for destruction by the 26S proteasome, changing its subcellular location, or changing its protein-protein interactions (Merkle, 2009).
Ubiquitination is a highly dynamic, multi-step process that requires three components: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2 or Ubc) and ubiquitin ligase (E3). E3s can be divided into two main classes: HECT and RING domain-containing proteins. RING-type E3 ubiquitin ligases contain a specialized motif of 40 to 60 residues that binds two zinc atoms. Many RING-type E3s bind to partnering E2 conjugating enzymes via their RING domains. Database searches of the Drosophila genome predict that it contains one E1, 36 E2s and ~130 E3s, which represents ~40% of the ubiquitination machinery in humans (Merkle, 2009).
Significant insights into the roles of many cell-cycle regulators have come from studying their functions in Drosophila. Drosophila is well suited for studying cell-cycle regulation during the formation of a multicellular organism, in large part because of its developmental use of cell cycles that differ in structure from canonical G1-S-G2-M cycles and the availability of genetic tools. The first thirteen cell cycles of Drosophila embryogenesis involve nearly synchronous nuclear divisions driven by stockpiles of maternally expressed mRNA and protein. These rapid cycles (~10 minutes in length) consist of oscillating S-M (DNA replication-mitosis) phases without intervening gap phases or cytokinesis. Minimal gene transcription occurs during this developmental stage, so cell cycles are regulated by post-transcriptional mechanisms. At cycle 14, the embryo cellularizes and initiates zygotic transcription at the midblastula transition (MBT) (Merkle, 2009).
This study reports the identification and characterization of a Drosophila maternal-effect lethal mutant 'no poles' (nopo). Embryos from nopo females undergo mitotic arrest with acentrosomal, barrel-shaped spindles during syncytial divisions. The results indicate that this arrest is secondary to the activation of a CHK2-mediated DNA checkpoint in early embryos. Nopo, a predicted E3 ubiquitin ligase, interacts with an E2 component, Ben. ben females are sterile, producing embryos with nopo-like defects. It is proposed that Ben-Uev1A and Nopo function together as an E2-E3 complex required for genomic integrity during Drosophila embryogenesis (Merkle, 2009).
nopo encodes a predicted protein of 435 amino acids containing an N-terminal RING domain. The putative mammalian homolog of Nopo was named 'TRAF-interacting protein' (TRIP) based on its ability to bind tumor necrosis factor (TNF) receptor-associated factors (TRAFs). Mammalian TRIP was recently demonstrated to have RING-dependent E3 ubiquitin ligase activity in an auto-ubiquitination assay. Drosophila Nopo and human TRIP are 20% identical and 34% similar overall, with 47% identity and 65% similarity in their RING domains. Importantly, nopoZ1447 causes a glutamic acid to lysine change in the RING domain at position 11 of the predicted protein, a residue that is invariantly negatively charged across species (Merkle, 2009).
A model is proposed in which Nopo interacts with the Ben-Uev1A heterodimer to form a functional E2-E3 ubiquitin ligase complex required during syncytial embryogenesis for genomic integrity, cell-cycle progression, and the continuation of development. In the absence of Nopo, a lack of ubiquitination of, as yet unidentified, Nopo targets results in the truncation of S-phase and/or spontaneous DNA damage. Mitotic entry with unreplicated and/or damaged DNA triggers the activation of a CHK2-mediated checkpoint that leads to changes in spindle morphology, mitotic arrest and failure of nopo-derived embryos to develop to cellularization (Merkle, 2009).
The idea is favored thatNopo regulates the timing of S-M transitions in syncytial embryos to ensure that S-phase is of sufficient length to allow the completion of DNA replication prior to mitotic entry. The inhibition of DNA replication in syncytial embryos (e.g. via aphidicolin injection) leads to chromatin bridging in subsequent mitoses and CHK2 activation, both of which occur in nopo-derived embryos, presumably because of mitotic entry with unreplicated chromosomes. The mechanism by which Nopo coordinates S-M transitions is unknown. The data suggest that nopo may alter the timing of these transitions independently of CDK1-Cyclin B, although localized changes in the levels and/or activities of these regulators not detectable by immunoblotting of whole-embryo lysates could play a crucial role. It is unclear why the MEI-41/GRP-dependent checkpoint, which appears to be functional in nopo-derived embryos, is not sufficient to slow mitotic entry (Merkle, 2009).
The punctate nuclear localization observed for Nopo and its human homolog, TRIP, expressed in HeLa cells may indicate a direct role for these proteins in the regulation of chromatin structure. Furthermore, the G2 phase-specific localization that was observe for Nopo/TRIP in transfected HeLa cells may be consistent with a role for Nopo in slowing S-M transitions in syncytial embryos; in the absence of nopo, embryos that enter mitosis prematurely would probably do so without finishing DNA replication because of a lack of gap phases (Merkle, 2009).
An alternative explanation for CHK2 activation in nopo-derived embryos is that they might incur elevated levels of spontaneous DNA damage. Syncytial embryos are considered to be unusual in that they activate CHK2 but not CHK1 in response to DNA-damaging agents. Thus, spontaneous DNA damage would not be predicted to elicit the MEI-41/GRP-mediated replication checkpoint but would cause CHK2-dependent centrosomal inactivation during mitosis. Such a model would be consistent with the apparent lack of activation of the MEI-41/GRP-dependent checkpoint in nopo-derived embryos, although it would not explain why interphase 11 is shortened (Merkle, 2009).
Syncytial embryos from microcephalin (mcph1) mutant females undergo mitotic arrest with a phenotype similar to that described for nopo (Rickmyre, 2007). Like nopo, CHK2-mediated centrosomal inactivation causes mitotic arrest in embryos lacking mcph1. nopo and mcph1 are unique among maternal-effect lethal mutants in which CHK2-mediated centrosomal inactivation has been reported (e.g., grp, mei-41, wee1) in that their phenotypes appear to be more severe: centrosomes typically detach from spindles, and mitotic arrest occurs earlier, during precortical syncytial divisions. The underlying defects in nopo and mcph1 mutants may be distinct, however, because mnk mcph1-derived embryos (referring to maternal nuclear kinase (mnk), also known as loki, which encodes Drosophila CHK2) exhibit normal cycle 11 interphase length, which is truncated in mnk nopo-derived embryos (Rickmyre, 2007). Furthermore, no genetic interaction was detected between nopo and mcph1 (Merkle, 2009).
Mammalian TRIP was identified in a yeast two-hybrid screen for tumor necrosis factor (TNF) receptor-associated factor (TRAF) interactors. TRAFs transduce signals from members of the tumor necrosis factor (TNF)/tumor necrosis factor receptor (TNFR) superfamily, which elicit diverse cellular responses in the immune and inflammatory systems. TRIP has been reported to inhibit TRAF2-mediated NFkappaB activation; the RING domain of TRIP, however, was not required for inhibition. By contrast, the current analysis of nopoZ1447 indicates that this motif is essential for Nopo function in Drosophila embryogenesis, probably by mediating its interactions with E2 components, as has been shown for other E3 ligases. Drosophila Eiger (TNF ligand) and Wengen (TNF receptor) play roles in dorsal closure, neuroblast divisions, and the response to fungal pathogens. A role for TNF signaling in early Drosophila embryogenesis has not been reported (Merkle, 2009).
TRIP was recently reported to be an essential factor in mice. TRIP-deficient mice die soon after implantation as a result of defects in early embryonic development. Compared with wild-type littermates, TRIP-/- embryos are smaller in size with a reduced cell number. TRAF2 does not appear to be required until later in development, suggesting that TRIP has TRAF2-independent roles in early embryos. It will be interesting to see whether mammalian TRIP, by analogy to Drosophila Nopo, is required for genomic integrity during embryonic development (Merkle, 2009).
The data support a model in which Nopo ubiquitin ligase acts in concert with Ben-Uev1A heterodimers to regulate Drosophila syncytial embryogenesis. The yeast two-hybrid interaction and co-localization of Nopo and Ben led to the identification of an unanticipated role for Ben in early embryogenesis and additional roles for Nopo in synapse formation and innate immunity. Although the spindle defects of ben-derived embryos are strikingly similar to those of nopo mutants, they typically arrest earlier in syncytial development, suggesting that another E3 ligase that requires Ben may function in parallel with Nopo. Although nopo egg chambers appear normal, a possible requirement for Ben-Uev1A-Nopo complexes during oogenesis has not been ruled out; some defects in nopo- and ben-derived embryos could be a secondary consequence of previous defects during oogenesis (Merkle, 2009).
K63-linked ubiquitin chains are thought to act as non-proteolytic signals (e.g. affecting protein localization and/or interactions), whereas K48-linked ubiquitin chains have established roles in targeting proteins for proteasome-mediated degradation. Ben-Uev1A E2 homologs in budding yeast (Ubc13-Mms2p) mediate K63-linked polyubiquitination of PCNA during postreplicative repair. In mammalian cells, the E2 heterodimer Ubc13-Mms2 mediates DNA damage repair, while Ubc13-Uev1A promotes NFkappaB activation; both E2 complexes regulate these processes by mediating K63 ubiquitin chain assembly on target proteins. It is proposed that Ben-Uev1A-Nopo (E2-E3) complexes mediate the assembly of K63-linked ubiquitin chains on proteins that preserve genomic integrity in early Drosophila embryogenesis (Merkle, 2009).
The checkpoint proteins, Rad9, Rad1, and Hus1 (9-1-1), form a complex which plays a central role in the DNA damage-induced checkpoint response. Drosophila hus1 has been shown to be essential for activation of the meiotic checkpoint elicited in double-strand DNA break (DSB) repair enzyme mutants. The hus1 mutant exhibits similar oocyte nuclear defects as those produced by mutations in these repair enzymes, suggesting that hus1 plays a role independent of its meiotic checkpoint activity. This study further analyzed the function of hus1 during meiosis. The synaptonemal complex (SC) was found to disassemble abnormally in hus1 mutants. Oocyte nuclear and SC defects of hus1 mutants can be suppressed by blocking the formation of DSBs, implying that the hus1 oocyte nuclear defects depend upon DSBs. Interestingly, eliminating checkpoint activity through mutations in DmChk2 but not mei-41 suppress the oocyte nucleus and SC defects of hus1, suggesting that these processes are dependent upon DmChk2 checkpoint activity. Moreover, in hus1, DSBs that form during meiosis are not processed efficiently, and this defect is not suppressed by a mutation in DmChk2. A genetic interaction was found between hus1 and the Drosophila brca2 homologue, which was shown to participate in DNA repair during meiosis. Together, these results imply that hus1 is required for repair of DSBs during meiotic recombination (Peretz, 2009)
When the integrity of the genetic material is compromised, the cell activates checkpoints that inhibit cell cycle progression, allowing for repair of the damaged DNA or, if unsuccessful, lead to cell death. The DNA damage checkpoint response involves a signal transduction pathway consisting of sensors, transducers and effectors. Hus1, Rad1 and Rad9 and the associated protein, Rad17 are thought to act as a sensor complex. The signal is transduced by ATM and ATM-Rad3-related (ATR) proteins along with Chk1 and Chk2 kinases. A wide range of effector proteins influence cellular fate following the DNA damage, among these are cell cycle arrest, apoptosis or activation of the DNA repair machinery. Various checkpoints exist, with each addressing a different type of DNA damage through the use of a specific set of signal transduction proteins (Peretz, 2009)
A meiotic recombination checkpoint, also known as the 'pachytene checkpoint' has been characterized in yeast. Meiotic recombination initiates with the generation of DNA double-strand breaks (DSBs) by the Spo11 endonuclease. These breaks are repaired via homologous strand exchange with sequences on a non-sister chromatid. A set of proteins monitors recombination and activates a checkpoint during late prophase I (pachytene) if the recombination repair process has not been completed. This checkpoint prevents segregation of homologous chromosomes until recombination is complete and ensures proper distribution of the genetic material to the gametes (Peretz, 2009)
A meiotic checkpoint similar to that described in yeast also exists in Drosophila. Several of the spindle class genes were previously found to encode proteins with homology to known DNA repair enzymes. Specifically, spindle-A (spn-A) encodes a Rad51-like protein, spindle-B (spn-B) encodes a XRCC3-like protein, spindle-C (spn-C) encodes a HEL308-like protein, spindle-D (spn-D) encodes a Rad51C-like protein and okra encodes a Rad54-like protein. These genes were shown to be required for the repair of recombination-induced DSBs during Drosophila oogenesis. Moreover, mutations in these genes lead to activation of a meiotic checkpoint, leading to the appearance of several defects during oogenesis. The most obvious phenotypes manifested are the dorsal-ventral (D-V) patterning defects of the egg, arising due to improper localization and translation of gurken mRNA. In addition, the hollow sphere of highly packed chromatin (also called the karyosome) that is characteristic of the wild-type oocyte nucleus is often fragmented or thread-like in appearance in the DNA repair enzyme mutants. These defects can be suppressed by blocking the formation of DSBs during meiosis through mutations in the spo11 homologue, mei-W68, or by eliminating the checkpoint through a mutations in mei-41 or DmChk2, the Drosophila homologues for ATR and Chk2, respectively (Peretz, 2009)
hus1 mutant flies have been shown to be viable although the females are sterile. hus1 mutant flies are sensitive to hydroxyurea (HU) and to methyl methanesulfonate (MMS) but not to X-rays, suggesting that hus1 is required for the activation of an S phase checkpoint. Furthermore, hus1 is not required for the G2/M checkpoint or for post-irradiation induction of apoptosis. hus1 is able to suppress the D-V pattering defects caused by mutations in DNA repair enzymes. Interestingly, hus1 mutants are also characterized by a range of karyosome formation defects, much like mutants expressing defective DNA repair enzymes. These results suggested that during meiosis, hus1 is required for efficient activation of the meiotic checkpoint in response to persistent DSBs and is also essential for the organization of the oocyte DNA, a function that may be independent of the meiotic checkpoint (Peretz, 2009)
This study further analyzes the role of hus1 during meiosis; hus1 was found to be required for the efficient repair of DSBs during homologous recombination (HR) in meiosis. hus1 genetically interacts with brca2. It was also shown that non-repaired DSBs in the hus1 mutant lead to activation of a DmChk2 checkpoint. These results thus suggest that hus1 plays a role in the repair of meiotic DSBs (Peretz, 2009)
This study shows that the aberrant karyosome phenotype in the hus1 mutant is caused by defective homologous recombination (HR) repair. Histone γ-His2Av phosphorylation, a DSB marker, was dramatically increased in hus1 mutant flies and these persisted until later stages of oogenesis, as compared to wild-type flies. Additionally, blocking the formation of DSBs by using mei-W68 mutant flies suppressed the karyosome defect of hus1 mutant. The persistence of DSBs and karyosome defects in hus1 mutants resemble phenotypes found in flies with mutations in DNA repair enzymes of the spindle class genes. Taken together, these findings suggest that hus1 functions not only in activating the meiotic (pachytene) checkpoint but also in the repair of DSBs by HR during meiosis. Supportive of a role for hus1 in HR is the finding that reducing hus1expression in mouse cells by a siRNA approach decreases the efficiency of HR repair (Wang, 2006). Mammalian Rad9, a member of the Rad9-Hus1-Rad1 complex (9-1-1), interacts with Rad51, and inactivation of mammalian Rad9 results in decreased HR repair (Pandita, 2006). In yeast cells, it was shown that Rad17, the Rad9 homologue, and Rad24, the Rad17 homologue, are required for repair of DSBs during meiosis by facilitating proper assembly of the meiotic recombination complex containing Rad51, a protein which catalyzes DNA strand invasion. Therefore, it seems likely that the 9-1-1 complex as a whole could function during HR, this requires further examining (Peretz, 2009)
Interestingly, flies mutant for the recently identified Drosophila brca2 gene, are characterized by a highly penetrant karyosome defect, weakly ventralized eggs and persisting DBSs, implying a role for brca2 in homologous recombination repair. The abnormal D-V eggshell phenotype in mutants of DNA repair enzymes can be suppressed by mutations in brca2. This suggests that brca2 plays an additional role in transduction of the meiotic recombination checkpoint signal (Klovstad, 2008). It was reasoned that such a requirement for brca2 in activation of the checkpoint masks the strong eggshell ventralization phenotype normally characteristic of mutants of DNA repair enzymes (Klovstad, 2008). A similar rational could be applied to results with the hus1 mutant, where hus1 represents another protein with a dual function in both DNA repair and checkpoint activation during Drosophila meiosis. It is suggested that hus1 and brca2 thus represent a new class of proteins that serve a dual function in HR repair and in checkpoint activation during meiosis but whose mutant alleles do not show the full and/or strong repertoire of phenotypes of classic repair enzyme mutants (Klovstad, 2008). Interestingly, it was also reported that the Drosophila ATR homologue, mei-41, serves a dual function in DNA damage checkpoint and in facilitating the later stages of HR repair. mei-41 mutants also show a pattern of γ-His2Av staining in oocytes similar to that seen in DSB repair mutants, including delayed onset and persistence of foci into late pachytene (Joyce, 2009). The reduced or partial phenotypes displayed in mutants of these proteins (hus1, brca2 and mei-41), as compared to DNA repair enzyme mutants of the spindle class, may be indicative of a more regulatory natured role in repair, rather than a direct one. Thus, in DNA repair mutants a meiotic checkpoint is activated due to lack in repair of DSBs, while in mutants of regulatory genes (such as hus1, brca2 and mei-41) DSBs are also not repaired, however the checkpoint is not transduced properly leading to less pronounced phenotypes (Peretz, 2009)
It was also found that a mutation in the DmChk2 gene was able to suppress the karyosome and SC disassembly defects observed in hus1 mutant egg chambers, although DSBs persisted in these double mutants. This implies that in flies lacking hus1, DSBs are not repaired and this, in turn, leads to the activation of a DmChk2-dependent checkpoint. A mei-41 mutation was, however, unable to suppress these karyosome and SC defects. Similar results were reported for brca2 mutants, where the karyosome defects were attributed to activation of DmChk2 checkpoint but not of mei-41dependent one (Klovstad, 2008). Supporting evidence to the inability of mei-41 to suppress hus1 karyosome defects is the finding that mei-41 (Joyce, 2009) but not DmChk2 (this study) mutants show defects in processing DSBs during meiosis. The activation of a DmChk2-dependent checkpoint in hus1 mutants could be due to activation of DmChk2 by the other upstream checkpoint kinase, ATM. At this point, using the karyosome suppression assay, it was not possible to test whether ATM is the upstream activator, since atm mutants themselves present karyosome defects. It will be interesting to test whether atm, as hus1 and brca2, has a dual role in activation of the meiotic checkpoint and in HR repair (Peretz, 2009)
Since Brca2 co-immunoprecipitated Rad9, a member of the 9-1-1 complex, (Klovstad, 2008) and this study demonstrated a dual function for hus1 in meiosis, which was similar to that of brca2, it was decided to test whether hus1 and brca2 genetically interact. Defects in oocyte localization and determination, which were found to be characteristic of other DNA repair enzymes, were used as an indirect outcome of DSB repair in the oocyte. A mutation in brca2 was shown to strongly enhance the oocyte localization and determination defects found in the hus1 mutants. The finding that both hus1 and brca2 mutants show defects in DSB repair in the oocyte and that Brca2 physically interacts with Rad9, a part of the 9-1-1 complex (Klovstad, 2008), suggest that hus1 and brca2 may be a part of the same pathway of HR repair (Peretz, 2009)
However, the genetic interaction between hus1 and brca2 in HR repair in the germarium pro-nurse cells could be interpreted in a different manner. In wild-type, in region 2a of the germarium some of the pro-nurse cells contained DSBs as revealed by γ-His2Av staining. In later stages of meiosis, region 2b-3, the DSBs were restricted only to the oocyte, suggesting that there is a mechanism that ensures the restriction of DSBs to the oocyte and prevents these breaks in pro-nurse cells. It was found that in the double mutant flies for hus1 and brca2, but not in the single mutants, all of the germaria nurse cells showed γ-His2Av staining, suggesting that the nurse cells throughout the germarium have DSBs. These results point towards the role of hus1 and brca2 in DSB repair both in the pro-nurse cells and the oocyte. Such defects in DSB repair in the nurse cells and the oocyte could be the cause for the apoptosis of egg chambers in brca2;hus1 double mutant flies. The finding that the defects in DSB repair in the pro-nurse cells were detected only in the double brca2;hus1 mutants but not in the single ones, suggests that in this process hus1 and brca2 could act in parallel or in redundant pathways. Since it was shown that mei-41 mutants do cause a persistence of γ-His2Av foci in the oocyte but not in the nurse cells (as in the hus1 mutant), it will be interesting to study the complex interactions between mei-4, brca2 and hus1 in this process (Joyce, 2009). Altogether, these results lead to the identification of Hus1 as a protein with a dual role in activation of the meiotic checkpoint and in HR repair during meiosis (Peretz, 2009)
Cyclin G (CycG) belongs to the atypical cyclins, which have diverse cellular functions. The two mammalian CycG genes, CycG1 and CycG2, regulate the cell cycle in response to cell stress. Detailed analyses of the role of the single Drosophila cycG gene have been hampered by the lack of a mutant. A null mutant was generated in the Drosophila cycG gene that is female sterile and produces ventralised eggs. This phenotype is typical of the downregulation of epidermal growth factor receptor (EGFR) signalling during oogenesis. Ventralised eggs are also observed in mutants (for example, mutants of the spindle class) that are defective in meiotic DNA double-strand break repair. Double-strand breaks (DSBs) induce a meiotic checkpoint by activating Mei-41 kinase (the Drosophila ATR homologue), thereby indirectly causing dorsoventral patterning defects. Evidence is provided for the role of CycG in meiotic checkpoint control. The increased incidence of DSBs in cycG mutant germaria may reflect inefficient DSB repair. Therefore, the downregulation of Mei-W68 (an endonuclease that induces meiotic DSBs), Mei-41, or Drosophila melanogaster Chk2 (a downstream kinase that initiates the meiotic checkpoint) rescues the cycG mutant eggshell phenotype. In vivo, CycG associates with Rad9 and BRCA2. These two proteins are components of the 9-1-1 complex, which is involved in sensing DSBs and in activating meiotic checkpoint control. Therefore, it is proposed that CycG has a role in an early step of meiotic recombination repair, thereby affecting EGFR-mediated patterning processes during oogenesis (Nagel, 2012).
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date revised: 15 April 2019
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