grapes


REGULATION

ATM and ATR pathways signal alternative splicing of Drosophila TAF1 pre-mRNA in response to DNA damage

Alternative pre-mRNA splicing is a major mechanism utilized by eukaryotic organisms to expand their protein-coding capacity. To examine the role of cell signaling in regulating alternative splicing, the splicing of the Drosophila TAF1 pre-mRNA was analyzed. TAF1 encodes a subunit of TFIID, which is broadly required for RNA polymerase II transcription. TAF1 alternative splicing generates four mRNAs, TAF1-1, TAF1-2, TAF1-3, and TAF1-4, of which TAF1-2 and TAF1-4 encode proteins that directly bind DNA through AT hooks. TAF1 alternative splicing was regulated in a tissue-specific manner and in response to DNA damage induced by ionizing radiation or camptothecin. Pharmacological inhibitors and RNA interference were used to demonstrate that ionizing-radiation-induced upregulation of TAF1-3 and TAF1-4 splicing in S2 cells is mediated by the ATM (ataxia-telangiectasia mutated) DNA damage response kinase and checkpoint kinase 2 (CHK2), a known ATM substrate. Similarly, camptothecin-induced upregulation of TAF1-3 and TAF1-4 splicing is mediated by ATR (ATM-RAD3 related) and CHK1. These findings suggest that inducible TAF1 alternative splicing is a mechanism to regulate transcription in response to developmental or DNA damage signals and provide the first evidence that the ATM/CHK2 and ATR/CHK1 signaling pathways control gene expression by regulating alternative splicing (Katzenberger, 2006; Full text of article).

Onset of the DNA replication checkpoint in the early Drosophila embryo

The Drosophila embryo is a promising model for isolating gene products that coordinate S phase and mitosis. Increasing maternal Cyclin B dosage to up to six copies (six cycB) increases Cdk1-Cyclin B (CycB) levels and activity in the embryo, delays nuclear migration at cycle 10, and produces abnormal nuclei at cycle 14. The level of CycB in the embryo inversely correlates with the ability to lengthen interphase as the embryo transits from preblastoderm to blastoderm stages and defines the onset of a checkpoint that regulates mitosis when DNA replication is blocked with aphidicolin. A screen for modifiers of the six cycB phenotypes identified 10 new suppressor deficiencies. In addition, heterozygote dRPA2 (a DNA replication gene) mutants suppressed only the abnormal nuclear phenotype at cycle 14. Reduction of dRPA2 also restored interphase duration and checkpoint efficacy to control levels. It is proposed that lowered dRPA2 levels activate Grp/Chk1 to counteract excess Cdk1-CycB activity and restore interphase duration and the ability to block mitosis in response to aphidicolin. These results suggest an antagonistic interaction between DNA replication checkpoint activation and Cdk1-CycB activity during the transition from preblastoderm to blastoderm cycles (Crest, 2007).

It has been proposed (model 1) that the ability of the DNA replication checkpoint to regulate the entry into M-phase is not active before cycle 11 in wild-type (two cycB) embryos. This model is based on observations that the earliest defects in interphase duration can be detected only after cycle 10 in grp (dChk1) or mei-41 (dATR) mutant embryos. In this view, before cycle 10 in two cycB embryos, nuclei have normal morphology because the DNA replication machinery is abundant. The exponentially increasing numbers of nuclei, however, titrate components of this machinery to a critical level after cycle 10, thereby inducing the checkpoint activation, interphase extension, and delay of M-phase in a Grp- and Mei-41-dependent manner (Crest, 2007).

An alternative (model 2) is proposed: Checkpoint function is active all the time, but before cycle 10 checkpoint activity is too low and is overridden by the high level of maternal Cdk1-CycB. The difference between the two models is that in model 1, checkpoint is activated by a critical amount of the replication machinery. In model 2, at a critical concentration of Cdk1-CycB, this kinase (Cdk1) can no longer override checkpoint activity. Several observations are not compatible with model 1, but are with model 2. First, here this study shows that checkpoint activity does not depend on a specific number of nuclei, number of rounds of divisions, or time after fertilization, but on the amount of CycB-Cdk1. It occurs earlier in one cycB embryos with fewer nuclei and later in six cycB embryos with more nuclei (Crest, 2007).

Second, model 1 does not explain interphase extension before cycle 10 and the slight increase of interphase even in grp mutant embryos after cycle 10 (Crest, 2007).

Third, Grp protein is required for the degradation of cyclin A in the presence of cycloheximide as early as cycle 4. Although the effect of Grp on CycA degradation may be different from its effect on replication checkpoint activation, this observation suggests that Grp is present and functional before cycle 10. How then do nuclei enter M-phase at the normal time when aph. is applied before cycle 10, i.e., show no DNA replication checkpoint? It is reasoned that successful execution of the checkpoint requires the inhibitory effect of Grp to overcome the M-phase-promoting effect of Cdk1-CycB. Early embryos might have too few nuclei, thus limited numbers of replication forks, to trigger sufficient Grp/Chk1 activity necessary to overcome the relatively high levels of Cdk1-CycB. Despite this situation, nuclear morphology is normal because replication machinery is not limited before cycle 10, S-phase is rapidly completed, and nuclei can successfully undergo a normal mitosis (Crest, 2007).

Fourth, during normal S-phase of either yeast or mammalian cells, a low level of replication checkpoint activity is observed. This low level of checkpoint activity can be detected as phosphorylation on Chk1 in S-phase cells without any replication stress or DNA damage. Physiological regulation of Chk1 is under the control of similar factors of the DNA replication checkpoint machinery, and thus it was proposed that DNA replication per se generates lesions that activated the checkpoint pathway. Alternatively, but not mutually exclusively, this constitutively low level of replication checkpoint activation may be due to transient signaling from the replication forks, which does not lead to cell cycle arrest, but serves as a mechanism to coordinate the firing of replication origins, thereby moderating the rate of S-phase (Crest, 2007).

Model 2 not only accounts for observations with the aph. experiments, but also accounts for observation of the CycB effects on the replication checkpoint effect. In one cycB embryos the Grp/Chk1 effect is observed earlier, presumably because levels of Cdk1-CycB become limiting earlier, whereas in six cycB embryos, these occur later. Using PH3 staining on anaphase chromosomes as a measurement for Chk1-dependent Cyclin A degradation indicates that Chk1 is not functioning in six cycB embryos before cycle 11, possibly because it is overridden by the abundance of Cdk1-CycB in these embryos. It is proposed that in six cycB embryos at cycle 11 or later, Cdk1-CycB activity is still too high and forces the nuclei into mitosis at a time when the DNA replication machinery is limited, resulting in precocious M-phase and abnormal nuclei (Crest, 2007).

This study addresses how dRPA2 might suppress the six cycB phenotype at cycle 14. dRPA2 is a subunit of a highly conserved heterotrimeric complex of proteins that make up the RPA complex. All three subunits contain DNA-binding domains, which stabilize ssDNA as it is unwound at the replication fork. This stabilized DNA allows for Cdc45 and DNA polymerase-α to initiate DNA replication. Additional roles for RPA have been implicated in DNA repair and recombination (Crest, 2007).

In six cycB embryos, during the blastoderm cycles, elevated levels of Cdk1-CycB override Chk1 and the nuclei divide before DNA replication is completed, leading to abnormal nuclei. It is speculated that reducing dRPA2 is likely to slow DNA replication because RPA can facilitate DNA replication by unwinding dsDNA and by modulating the activities of several enzymes, such as DNA helicases, DNA polymerases, and primases. This would result in less RPA coated, primed DNA and ssDNA. Such a DNA structure may potentiate the TopBP1-mediated ATR-ATRIP kinase activation, leading to stronger Chk1 activation. Thus in dRPA2/six cycB embryos, a stronger Chk1 activation would have a stronger inhibitory effect on Cdk1-CycB activity that cancels out the effect caused by extra Cdk1-CycB. This interpretation for the suppressive effect of RPA2 on the six cycB phenotype suggests an antagonistic relationship between the DNA replication checkpoint activation and Cdk1-CycB activity in regulating the transition from the preblastoderm cycles to the blastoderm cycles (Crest, 2007).

The earliest divisions in the embryo (with the exception of mammals) are maternally regulated until the zygotic genome takes over. On the basis of many observations with different animals such as Xenopus and Drosophila, a simple concept developed: Early cell cycles are invariant, synchronous, and lack both gap phases until the transition to zygotic control occurs. The time point at which this change happens has been called the midblastula transition (MBT). It is assigned to the specific cycle when zygotic transcription is activated. Furthermore, G2 is induced in Drosophila and G1 and G2 in Xenopus into the abbreviated cell cycles and cell division is patterned (Crest, 2007).

This concept is attractive; however, as with many simple concepts, the more that is learned the harder it is to accept the concept at face value. For example, synchronous divisions have never been observed in Caenorhabditis elegans. Even in Drosophila, the simplification that early cell cycles are synchronous and equal in length is incorrect since interphase durations steadily increase after cycle 7 and metasynchronous mitoses are observed as early as cycle 4. These changes occur long before cycle 14, the time that has been designated by many as the MBT. The data presented in this study clearly demonstrate a change in the maternal program as the embryo develops: A DNA replication checkpoint is first detectable after cycle 10, but becomes increasingly robust in the subsequent cycles, indicating that the ability to regulate M-phase by checkpoints is not completely'off' or 'on'. In addition, it was found that the DNA replication checkpoint is detectable earlier in one cycB and later in six cycB embryos, clearly indicating that changes do not have to occur at a specific stage. A gradual attainment of full checkpoint function is also supported by the fact that aph. injections before cycle 11 can stall/delay the nuclear cycle, but not the centrosomal cycle. These results are not compatible with the idea of an invariant maternal program for pre-MBT cycles (Crest, 2007).

Another simplification is that changes that define the MBT are events that envelope the entire embryo. In sea urchins the deceleration of the cell cycle in the micromeres occurs long before these changes have been observed in the macromeres. Thus an MBT has not been proposed for this animal. But differences are also observed among the blastoderm cycles in Drosophila, where divisions in the middle of the embryo are slower than at the poles, correlating with their nuclear densities (Crest, 2007).

The initiation of the zygotic program also does not occur suddenly from off to on. fushi tarazu (ftz) transcripts are first observed at cycle 9 in one or the other embryo and in one or the other nucleus. Transcription gradually increases over the next three cycles (cycles 9-12). This gradual increase is a consequence of the dose-dependent repressor Tramtrack (TTK), where with one gene dose of maternal ttk, initial transcription of ftz occurs one cycle earlier, and conversely extra copies of ttk result in initial transcription of ftz one cycle later. These data are interpreted as a decline of TTK during cycles 8-10 to a threshold level where TTK repression is insufficient, enabling low-level transcription of ftz (Crest, 2007).

Despite the many observations that do not fit the MBT concept, textbooks, reviews, research articles, and grant proposals still hold on to it, hindering progress in the understanding of how maternally controlled development declines and terminates and zygotic programming gradually takes over (Crest, 2007).

Protein Interactions

Both control and grapes-derived embryos, collected between 0 and 2.5 hours of development, predominantly exhibit two fast migrating forms of Cdc2; these correspond to the unmodified and the active Thr161-phosporylated phosphoisoforms. In control embryos collected between 2.5 and 5 hours (which include a significant fraction of nuclear cycle 14 embryos), two slower migrating Cdc2 isoforms are present. Previous studies have shown that these represent two inactive phosphoisoforms of Cdc2 (phosphorylated at Tyr15 and Thr14, or Tyr15 only) that accumulate to high levels during the interphase of cycle 14. Traces of the Tyr15-phosphorylated inactive Cdc2 isoform seen in the control 0-2.5 hour sample can be accounted for by a small proportion of older embryos present in these collections. Intriguingly, the slowest migrating inhibitory phosphoisoform of Cdc2 (phosphorylated at Thr14 and Tyr15) never appears in grp-derived embryos collected between 2.5 and 5 hours, and the accumulation of the Tyr15-phosphorylated inactive phosphoisoform is greatly reduced (Fogarty, 1997).


DEVELOPMENTAL BIOLOGY

Embryonic

Throughout the first 12 nuclear cycles, maternally provided GRP mRNA is abundant and homogeneously distributed. As the embryo progresses from nuclear cycle 12 to 13, extensive degradation of the maternal GRP mRNA occurs, with the remaining transcripts assuming a pronounced anterior and posterior localization. Whether this pattern reflects a spatial difference in the kinetics of degradation or a difference in the initial abundance of GRP mRNA is not known. From early interphase of nuclear cycle 14 through cellularization, the remaining GRP mRNA is degraded (Fogarty, 1997).

Effects of Mutation or Deletion

Embryogenesis is typically initiated by a series of rapid mitotic divisions that are under maternal genetic control. The switch to zygotic control of embryogenesis at the midblastula transition is accompanied by significant increases in cell-cycle length and gene transcription, and changes in embryo morphology. Mutations in the grapes (grp) checkpoint 1 kinase homolog in Drosophila block the morphological and biochemical changes that accompany the midblastula transition, lead to a continuation of the maternal cell-cycle program, and disrupt DNA-replication checkpoint control of cell-cycle progression. The timing of the midblastula transition is controlled by the ratio of nuclei to cytoplasm (the nucleocytoplasmic ratio), suggesting that this developmental transition is triggered by titration of a maternal factor due to the increasing mass of nuclear material that accumulates during the rapid embryonic mitoses. These observations support a model for cell-cycle control at the midblastula transition in which titration of a maternal component of the DNA-replication machinery slows DNA synthesis and induces a checkpoint-dependent delay in cell-cycle progression. This delay may allow both completion of S phase and transcription of genes that initiate the switch to zygotic control of embryogenesis (Sibon, 1997).

Relative to normal embryos, embryos derived from grp-mutant mothers exhibit elevated levels of DNA damage. During nuclear cycles 12 and 13, the alignment of chromosomes on the metaphase plate is disrupted in grp-derived embryos, and the embryos undergo a progression of cytological events that are indistinguishable from those observed in normal syncytial embryos exposed to X-irradiation. The mutant embryos also fail to progress through a regulatory transition in Cdc2 activity, which normally occurs during interphase of nuclear cycle 14. It is proposed that the primary defect in grp-derived embryos is a failure to replicate or repair DNA completely before mitotic entry during the late syncytial divisions. This suggests that wild-type grp functions in a developmentally regulated DNA replication/damage checkpoint operate during the late syncytial divisions (Fogarty, 1997).

X-irradiation of embryos between nuclear cycles 9 and 11 results in an increase in cells in prometaphase relative to cells in metaphase, when examined 9 minutes after irradiation. This suggests that the initial response to DNA damage is a delay in the cell cycle during prometaphase. At 25 minutes after X-irradiation, the frequency of sister telophase fusion is much higher and a large percentage of embryos contain decondensed nuclei encompassed by multipolar and malformed spindles. Many of the nuclei fixed 25 minutes after X-irradiation contain a pair of centrosomes at each pole. This is due to fusion of sister telophase nuclei. At this point the nuclei accumulate in an abnormal multipolar metaphase. These defects are equivalent to those observed in unirradiated grapes-derived mutant embryos. These shared phenotypes suggest that, during the late syncytial divsions, unirradiated grp-derived embryos may also be entering mitoisis with damaged or incompletely replicated DNA. Incubation of isolated grapes-derived genomic DNA with T4 DNA kinase and labelled ATP (an assay that tests the number of exposed 5' phosphate groups in the DNA and thus the number of single stranded and double stranded lesions) reveals a dramatic increase in lesions as a result of grapes mutation. Alignment of chromosomes is also disrupted in grp-derived embryos (Fogarty, 1997).

grapes (grp) is a second chromosome (36A-B) maternal-effect lethal mutation in Drosophila melanogaster. The syncytial nuclear divisions of grp-derived embryos are normal through metaphase of nuclear cycle 12. However, as the embryos progress into telophase of cycle 12, the microtubule structures rapidly deteriorate and midbodies never form. Immediately following the failure of midbody formation, sister telophase products collide and form large tetraploid nuclei. These observations suggest that the function of the midbody in the syncytial embryo is to maintain separation of sister nuclei during telophase of the cortical divisions. After an abbreviated nuclear cycle 13 interphase, these polyploid nuclei progress through prophase and arrest in metaphase. The spindles associated with the arrested nuclei are stable for hours even though the microtubules are rapidly turning over. The nuclear cycle 13 anaphase separation of sister chromatids never occurs and the chromosomes, still encompassed by spindles, assume a telophase conformation. Eventually neighboring arrested spindles begin to associate and form large clusters of spindles and nuclei. To determine whether this arrest is the result of a disruption in normal developmental events that occur at this time, both grp-derived and wild-type embryos were exposed to X-irradiation. Syncytial wild-type embryos exhibit a high division error rate, but not a nuclear-cycle arrest after exposure to low doses of X-irradiation. In contrast, grp-derived embryos exhibit a metaphase arrest in response to equivalent doses of X-irradiation. This arrest can be induced even in the early syncytial divisions prior to nuclear migration. These results suggest that the nuclear cycle 13 metaphase arrest of unexposed grp-derived embryos is independent of the division-cycle transitions that also occur at this stage. Instead, it may be the result of a previously unidentified feedback mechanism (Fogarty, 1994).

Drosophila embryogenesis is initiated by 13 rapid syncytial mitotic divisions that do not require zygotic gene activity. This maternally directed cleavage phase of development terminates at the midblastula transition (MBT), at which point the cell cycle slows dramatically, membranes surround the cortical nuclei to form a cellular blastoderm, and zygotic gene expression is first required. Embryos lacking Mei-41, a Drosophila homolog of the ataxia telangiectasia (ATM) tumor suppressor, proceed through unusually short syncytial mitoses, fail to terminate syncytial division following mitosis 13, and degenerate without forming cells. Transcription of gap and pair rule genes show expected expression patterns during syncytial divisions 12 and 13, but on completion of mitosis 13 the expected high-level, spatially restricted expression of these genes is not initiated. Mutations in mei-41 thus prevent the onset of the high-level, patterned gene expression that characterizes the transition from maternal to zygotic control of development. It is thought that mei-41 has a specific role in zygotic gene activation (Sibon, 1999).

A similar cleavage-stage arrest is produced by mutations in grapes, which encodes a homolog of the Checkpoint-1 kinase. Biochemical, cytological and genetic data is presented indicating that Mei-41 and Grapes are components of a conserved DNA-replication/damage checkpoint pathway that triggers inhibitory phosphorylation of the Cdc2 kinase and mediates resistance to replication inhibitors and DNA-damaging agents. Double grapes:mei-41 mutants are indistinguishable from either single mutant, supporting the hypothesis that the two genes function in the same pathway. The grp null allele is a dominant enhancer of the mei-41 embryonic lethality, further supporting the hypothesis that Mei-41 and Grapes function in the same pathway during embryogenesis. The pathway utilizing Mei-41 and Grape is required to terminate the cleavage stage at the MBT. This pathway is nonessential during postembryonic development. Animals homozygous for null alleles of mei-41 and grp develop to the adult stage, are female-sterile, and produce embryos with normal external morphology that are arrested in the cleavage stage of embryogenesis. Cyclins are required for Cdc2 kinase activity, and mutations in cyclin A and cyclin B bypass the requirement for mei-41 at the MBT. Reduced cyclin A and cyclin B are likely to increase the cell cycle to an extent that allows production of an early zygotic factor that might be required at the MBT. Cyclin mutations do not restore wild-type syncytial cell-cycle timing or the embryonic replication checkpoint, however, suggesting that Mei-41-mediated inhibition of Cdc2 has an additional essential function at the MBT (Sibon, 1999).

The timing of the MBT is controlled by the nucleocytoplasmic ratio, suggesting that this transition, triggered as a result of the titration of a limiting factor by DNA or chromatin during the later cleavage divisions. On the basis of observations made in this study, a model is favored in which a maternal component of the DNA replication machinery serves as the titrated maternal factor that regulates the timing of the MBT. In this model, the replication machinery is in excess and the time required to complete the synthesis (S) phase of the cell cycle is constant until division 10, when at least one replication factor becomes limiting. After this point, the length of time required for chromosome replication progressively increases, and the checkpoint pathway is therefore required to delay mitosis to allow S-phase completion. This model explains the progressive increase in S-phase length during the syncytial-blastoderm divisions, and the requirement for repliction-checkpoint function for these increases. It is concluded that the Drosophila DNA-replication/damage checkpoint pathway can be activated by externally triggered DNA damage or replication defects throughout the life cycle, and under laboratory conditions this inducible function is nonessential for growth to adulthood. During early embryogenesis, however, this pathway is activated by developmental cues and is required for the transition from maternal to zygotic control of development at the MBT (Sibon, 1999).

The Drosophila grapes (grp) gene, which encodes a homolog of the Schizosaccharomyces pombe Chk1 kinase, provides a cell-cycle checkpoint that delays mitosis in response to inhibition of DNA replication. Grp is also required in the undisturbed early embryonic cycles: in its absence, mitotic abnormalities appear in cycle 12 and chromosomes fail to fully separate in subsequent cycles. In other systems, Chk1 kinase phosphorylates and suppresses the activity of Cdc25 phosphatase: the resulting failure to remove inhibitory phosphate from cyclin-dependent kinase 1 (Cdk1) prevents entry into mitosis. Because in Drosophila embryos Cdk1 lacks inhibitory phosphate during cycles 11- 13, it is not clear that known actions of Grp/Chk1 suffice in these cycles. It was found that the loss of grp compromises Cyclin A proteolysis and delays mitotic disjunction of sister chromosomes. These defects occur earlier than previously reported grp phenotypes. It is concluded that Grp activates Cyclin A degradation, and functions to time the disjunction of chromosomes in the early embryo. As Cyclin A destruction is required for sister chromosome separation: a failure in Grp-promoted cyclin destruction can also explain the mitotic phenotype. The mitotic failure described previously for cycle 12 grp embryos might be a more severe form of the phenotypes that are describe here in earlier embryos and it is suggested that the underlying defect is reduced degradation of Cyclin A (Su, 1999).

When syncytial embryos are exposed to cycloheximide, a protein synthesis inhibitor, nuclear cycles arrest in interphase and cyclin A levels decline whereas Cyclin B is stable under these conditions. This suggests that a steady state of synthesis and degradation maintains the interphase levels of Cyclin A during the syncytial divisions. grp mutants were found to be deficient in Cyclin A degradation in the presence of cycloheximide. This defect is seen in embryos in cycles 4- 8, earlier than other reported grp phenotypes and is therefore unlikely to be secondary to these phenotypes. It is inferred that Grp normally destabilizes Cyclin A (Su, 1999).

In wild-type embryos, Cyclin A is unstable not only in interphase but also during mitotic arrest caused by microtubule destabilization. Thus, in embryos treated with colchicine, blocking protein synthesis with cycloheximide leads to a decline in Cyclin A levels, whereas cyclin B is stable under these conditions. grp embryos are compromised for Cyclin A proteolysis at such a colchicine-induced mitotic arrest. As for interphase destruction of Cyclin A, this defect is seen during early syncytial cycles, before the onset of other reported grp phenotypes. It is concluded that Grp promotes Cyclin A degradation in colchicine-arrested early embryos (Su, 1999).

If normal levels of Cyclin A are maintained by a steady state of synthesis and destruction, it would be expected that the levels of Cyclin A would be high in grp embryos as a result of increased stability. Western blotting shows that grp embryos have slightly higher levels of Cyclin A than wild-type embryos (1.5-3-fold). Although this relatively small increase suggests that significant degradation of Cyclin A still occurs in grp embryos, this degradation is not apparent at the arrests induced by cycloheximide or by colchicine (Su, 1999).

Since Grp is required for destruction of Cyclin A in an arrested mitosis, it was determined whether or not mitosis is disrupted in grp embryos. The mitosis-specific phosphorylation of histone H3 (PH3) apparently acts as an in vivo reporter for cyclin/Cdk activity and its disappearance at the end of mitosis requires cyclin destruction. During syncytial cycles of wild-type embryos, PH3 staining is continuous along the length of the chromosome arms from metaphase until late anaphase, when loss of the epitope near kinetochores leads to graded staining. In embryos from grp1 homozygous females or from grp1/Df females, the gradient of PH3 is seen on chromosomes in early anaphase. Thus, PH3 loss is advanced with respect to chromosome segregation in grp embryos. This defect is seen at the earliest cycles scored (cycle 4 in grp1/grp1 and cycle 3 in grp1/Df), well before the onset of previously reported defects in grp embryos (cycle 11) (Su, 1999).

The loss of PH3 during early anaphase in grp embryos could be due to the premature loss of PH3, a scenario opposite of that expected for a mutation that stabilizes Cyclin A during mitosis. Alternatively, timely loss of PH3 staining but delayed chromosome separation would produce the same mis-coordination. Analysis of grp embryos supports the latter hypothesis: an increase in the ratio of embryos with unsegregated chromosomes (prophase/metaphase) to those with segregated chromosomes (anaphase/telophase) has been found. It ia concluded that Grp is required for timely chromosome segregation in syncytial mitoses (Su, 1999).

The mitotic phenotype in grp embryos can be understood as follows: ordinarily, the mitotic cyclins are degraded in a sequence during exit from mitosis; Cyclin A is degraded before the metaphase-anaphase transition; cyclin B is degraded at the beginning of anaphase and cyclin B3 towards the end of anaphase. The disappearance of PH3 can be prevented by stabilization of any of these mitotic cyclins; thus, it appears that loss of PH3 marks the completion of this sequence. It is suggested that grp embryos are specifically defective in the early initiation of Cyclin A degradation but they do degrade Cyclin A, perhaps in conjunction with the B cyclins. Eventual destruction of Cyclin A would explain the ability of grp-deficient nuclei to exit mitosis and lose PH3 staining, events that can be inhibited by stable Cyclin A. Destruction of Cyclin A in conjunction with the B cyclins would explain why the length of mitosis is not increased in grp embryos. Because the expression of a stable form of Cyclin A prevents chromosome disjunction, it has been suggested that the failure to degrade Cyclin A early during mitosis in grp embryos delays chromosome disjunction. The delay in chromosome separation abbreviates anaphase and, when the abbreviation is severe, decondensation of chromosomes and entry into the next interphase occurs before the separating chromosomes reach the spindle poles (Su, 1999).

Since Grp promotes Cyclin A degradation, genetic interactions might be detected between grp and Cyclin A. A test was performed to see whether grp mutants are sensitive to levels of Cyclin A. Homozygous grp flies are viable but female sterile. When a single copy of a heat-inducible Cyclin A transgene is introduced, however, homozygous grp1 progeny are not recovered. Thus, even without induction, the presence of a heat-inducible Cyclin A transgene (which by itself is viable as a heterozygote or homozygote) causes grp1 to behave as a recessive lethal. The observed synthetic lethality suggests that the Grp deficiency sensitizes the fly to low levels of Cyclin A expression from the transgene. In contrast to this strong interaction with increased Cyclin A levels, a reduction in the dose of Cyclin A fails to suppress the grp1 allele. It is perhaps not surprising that reduction of Cyclin A by half does not suppress a null allele of grp. Reduction in the dose of Cyclin A does suppress the lethality of mei-41 mutations. The mei-41 gene is a homolog of the gene ATM, which is mutated in the genetic disorder ataxia-telangiectasia; mei-41 is thought to act upstream of grp in the checkpoint pathway, and mutations in mei-41 result in a phenotype like grp, but less severe. Importantly, the suppression of the mei-41 embryonic lethality by cyclin reduction occurs without restoring interphase length. This result shows that the mitotic defect is not an inevitable consequence of premature entry into mitosis as previously thought. It is suggested that the mitotic defect is an anaphase failure as a result of defective metaphase destruction of Cyclin A (Su, 1999).

If Grp promotes the metaphase-anaphase transition, why is it dispensable at most stages of development? Before cell cycle 12, grp embryos exhibit a defective mitosis with delayed sister chromosome separation; despite this, mitosis is successful. From this observation, two inferences can be made: (1) mitosis can tolerate a limited disruption in the timing of events and, (2) as anaphase occurs in the absence of Grp, there must be a backup Grp-independent mechanism that promotes sister separation slightly later. The mitotic mis-coordination in grp embryos gets progressively more severe, and Cyclin A levels increase progressively during the syncytial cycles. This correlation, together with the ability of reduced Cyclin A dose to suppress mei-41 lethality, leads the authors to suggest that the consequence of a defect in the grp/mei-41 pathway increases in severity as Cyclin A increases, until anaphase fails at mitosis 12 and 13 (Su, 1999).

The current model for Chk1 function involves the phosphorylation and inhibition of Cdc25, in part by the binding of 14-3-3 protein to the phosphorylated Cdc25 and sequestration in the cytoplasm where it is ineffective in counteracting the nuclear kinases Wee1 and Mik1. Thus, inhibitory phosphorylation of Cdk1 prevents its activation and the cell arrests in G2. Although this action of Chk1 appears general, it is possible that Chk1 activity has other consequences. Indeed, there is no substantial accumulation of inhibitory phosphate on Cdk1, and the Cdc25Stg protein is constitutively present and nuclear during interphase of syncytial cycles 11- 13 when a grp-dependent mechanism regulates the entry into mitosis. The results presented here suggest that Grp may function to destabilize Cyclin A. When a Grp-dependent cell-cycle checkpoint is induced by blocking S phase with aphidicolin in cleavage-stage Drosophila embryos, Cdc25Stg is destabilized. Thus, whether it is direct or indirect, Grp promotes the destruction of two cell-cycle proteins, Cdc25Stg and Cyclin A. It is suggested that promotion of the metaphase- anaphase transition represents a second function of the grp/mei-41 pathway, distinct from the checkpoint arrest of entry into mitosis. Nevertheless, a common mechanism might be involved because blocking entry into mitosis and promoting exit from mitosis both involve inhibition of cyclin/Cdk1 activity (Su, 1999).

In summary, Grp is required for normal Cyclin A turnover in the early Drosophila embryo. It was also found that grp mutant embryos show a delay in the timing of the metaphase- anaphase transition. Stable versions of Cyclin A block chromosome separation at the metaphase plate, at least in cellularized Drosophila embryos, suggesting that proteolysis of Cyclin A is required for this process. Thus, the proposal that Grp activates Cyclin A proteolysis can explain the mitotic phenotype as a consequence of at least temporary persistence of Cyclin A (Su, 1999).

During early embryogenesis of Drosophila, mutations in the DNA-replication checkpoint lead to chromosome-segregation failures. These segregation failures are associated with the assembly of an anastral microtubule spindle, a mitosis-specific loss of centrosome function, and dissociation of several components of the gamma-tubulin ring complex from a core centrosomal structure. The DNA-replication inhibitor aphidicolin and DNA-damaging agents trigger identical mitotic defects in wild-type embryos, indicating that centrosome inactivation is a checkpoint-independent and mitosis-specific response to damaged or incompletely replicated DNA. It is proposed that centrosome inactivation is part of a damage-control system that blocks chromosome segregation when replication/damage checkpoint control fails (Sibon, 2000).

It has been proposed that a component of the DNA-synthesis machinery becomes rate-limiting during the later syncytial divisions, and that activation of the DNA-replication checkpoint thus produces the increases in embryonic cell-cycle length that characterize these divisions. This model predicts that checkpoint-mutant embryos will initiate mitosis before completing DNA replication specifically during the later syncytial divisions, when the mitotic defects first appear. It was therefore speculated that the centrosome defects and segregation failures in checkpoint-mutant embryos resulted from incomplete DNA replication at mitosis, and that the checkpoint pathway is not specifically required to maintain centrosome function. In wild-type embryos, the DNA-replication inhibitor aphidicolin triggers cell-cycle delays, but mitosis is eventually initiated before S phase is completed. Thus, to test the hypothesis, mitosis was examined following treatment of wild-type embryos with aphidicolin. Under these conditions, gamma-tubulin, and the gamma-tubulin ring complex components Dgrip84 and Dgrip91 are displaced from a core centrosome structure. Aphidicolin triggers mitosis-specific loss of centrosomal gamma-tubulin, Dgrip84 and Dgrip91 during all of the syncytial divisions, in both wild type and checkpoint mutants. This structural response to aphidicolin is therefore replication-checkpoint-independent and is not specific to the later syncytial divisions. This structural response to aphidicoline occurs in grp and mei-41 mutant embryos, and is therefore replication-checkpoint-independent (Sibon, 2000).

To analyse the effects of aphidicolin on spindle assembly and chromosome segregation directly, embryos were injected with a mixture of aphidicolin, rhodamine-labelled tubulin and the DNA label Oligreen and were examined by time-lapse confocal microscopy. The results showed that aphidicolin leads to loss of tubulin foci at nuclear envelope breakdown (NEB), assembly of anastral spindles, and defects in chromosome congression and segregation. Furthermore, the centrosomal foci are re-established on exit from mitosis. Thus aphidicolin treatment accurately phenocopies the mitotic defects observed in checkpoint mutants, indicating that centrosome inactivation and the associated chromosome-segregation failures in these mutants are probably triggered by the presence of incompletely replicated DNA at mitosis (Sibon, 2000).

To determine whether photodamage to DNA also triggers centrosome inactivation and chromosome-segregation defects, laser illumination was used to induce DNA damage. Bipolar spindles are established shortly after NEB, at the time that microtubules originating at the poles interact with each other and with the mitotic chromosomes, and chromosomes rapidly congress to the metaphase plate. During mitosis 12, anaphase is initiated roughly 4 min after NEB, and chromosome segregation is completed and the nuclear envelope begins to reform ~3 min later. However, when the intensity of laser illumination is increased to 90% of maximum, the centrosomal tubulin foci decrease in intensity at NEB and disorganized microtubule bundles form around the condensed chromosomes. Bipolar spindles with broad poles eventually assemble, but the chromosome arms never fully compact at the metaphase plate and chromosome segregation fails. On intense laser illumination in checkpoint mutants and in wild-type embryos, small tubulin-containing dots are often found near poles of the anastral spindles. These structures do not appear to nucleate microtubules during mitosis, but gain nucleating function on exit from mitosis. These structures thus appear to be centrosomes that are inactivated at NEB and reactivated on exit from mitosis. X-ray treatments and ultraviolet light also trigger mitosis-specific loss of centrosomal gamma-tubulin, anastral spindle assembly, and defects in chromosome congression and segregation. On the basis of these observations, it is concluded that a variety of DNA-damaging agents and replication defects can trigger mitosis-specific centrosome inactivation and mitotic chromosome-segregation defects (Sibon, 2000).

Thus it has been shown that the chromosome-segregation defects in grp and mei-41 checkpoint-mutant embryos are linked to loss of centrosome function and dissociation of several components of the gamma-tubulin ring complex from a core centrosomal structure. However, this does not reflect a specific requirement for grp or mei-41 in maintaining centrosome function. The alleles analysed here are null, yet the mutant embryos proceed through 11-12 normal mitotic divisions before mitotic defects are observed. In addition, replication inhibitors or DNA-damaging agents trigger cytologically identical centrosome defects in wild-type embryos. It is therefore concluded that the loss of centrosome function and mitotic failures in grp and mei-41 mutants reflect activation of a replication-checkpoint-independent response to incomplete DNA replication or DNA damage at mitosis (Sibon, 2000).

Incomplete DNA replication or DNA damage could lead to kinetochore defects, which trigger the spindle-assembly checkpoint; centrosome inactivation could be a response to activation of this checkpoint. However, the localization of gamma-tubulin to the centrosome is not significantly affected by treatment with colchicine, which triggers metaphase arrest through the spindle-assembly checkpoint pathway. In addition, centrosome inactivation does not block centromere alignment, indicating that kinetochore function is not severely compromised when the centrosomes are inactivated. Therefore, DNA-replication- and DNA-damage-associated centrosome inactivation are both independent of the replication and the spindle checkpoints, and appear to reflect the action of a new pathway that links centrosome function to the physical state of the genome (Sibon, 2000).

Mitotic spindle assembly is normally dominated by astral microtubules, which are nucleated at centrosomes. The chromosome-segregation defects in checkpoint mutants and in wild-type embryos treated with aphidicolin or DNA-damaging agents are tightly linked to loss of centrosome-organized microtubules. These observations indicate that asters may be required for spindle function. However, anastral spindles are common during female meiosis and are found in some unusual mitotic systems and cell-free extracts. In addition, spindles in Drosophila embryos mutant for cnn have severely reduced asters, and these anastral spindles may mediate chromosome segregation. Therefore, DNA replication or damage could induce centrosome inactivation and modifications to other functions that are essential for anastral spindle function (Sibon, 2000).

In Drosophila, the maternally expressed mei-41 and grapes genes are required for successful execution of the nuclear division cycles of early embryogenesis. In fission yeast, genes encoding similar kinases (rad3 and chk1, respectively) are components of a cell cycle checkpoint that delays mitosis by inhibitory phosphorylation of Cdk1. Mutations have been identified in Drosophila wee. Like mei-41 and grp, wee is zygotically dispensable but is required maternally for completing the embryonic nuclear cycles. The arrest phenotype of wee mutants, as well as genetic interactions between wee, grp, and mei-41 mutations, suggest that wee is functioning in the same regulatory pathway as these genes. These findings imply that inhibitory phosphorylation of Drosophila Cdk1 (alternatively termed Cdc2) by Wee is required for proper regulation of the early syncytial cycles of embryogenesis (Price, 2000).

Extra maternal copies of the genomic wee transgene were provided in a mei-41D3 mutant background. Females homozygous for the mei-41D3 allele produce cellularized embyros at a very low frequency (2%). The frequency of cellularized embryos is dramatically increased by adding an extra maternal copy of a wee genomic transgene (20%). The mei-41D3 mutant embryos are further rescued by addition of two wee transgenes (50%), to the extent that some mei-41D3-derived embryos are able to develop to adulthood. In contrast, parallel experiments in a grp1 background did not produce any rescue of the mutant phenotype with either one or two extra copies of wee. The simplest interpretation that can be offered for why the results differ between grp and mei-41 mutants in these experiments is that the mei-41D3 is not a complete loss-of-function allele, and consequently mei-41D3 mutants are more sensitive to increased dosage of wee than grp1 mutants. Alternatively, grp may respond to two different signaling pathways whereas mei-41 may respond to only one of the two. Wee1 overproduction could be sufficient to rescue the common function but not the grp-specific one according to this model. Another test for functional interactions among these genes was to assess the effect of lowering the maternal dosage of mei-41+ or grp+ in a homozygous Dwee1DS1 maternal background. The incompletely penetrant syncytial arrest phenotype of homozygous Dwee1DS1-derived embryos (54% cellularized) was enhanced by subtracting a maternal copy of mei-41+ (39%). Removal of one maternal copy of grp+ produced an even greater enhancement of the mutant phenotype of Dwee1DS1 embryos (29% cellularized) (Price, 2000).

The earliest embryonic mitoses in Drosophila, as in other animals except mammals, are viewed as synchronous and of equal duration. However, total cell-cycle length steadily increases after cycle 7, solely owing to the extension of interphase. Between cycle 7 and cycle 10, this extension is DNA-replication checkpoint independent, but correlates with the onset of Cyclin B oscillation. In addition, nuclei in the middle of embryos have longer metaphase and shorter anaphase than nuclei at the two polar regions. Interestingly, sister chromatids move faster in anaphase in the middle rather than the posterior region. These regional differences correlate with local differences in Cyclin B concentration. After cycle 10, interphase and total cycle duration of nuclei in the middle of the embryo are longer than at the poles. Because interphase also extends in checkpoint mutant (grapes) embryos after cycle 10, although less dramatic than wild-type embryos, interphase extension after cycle 10 is probably controlled by both Cyclin B limitation and the DNA-replication checkpoint (Ji, 2004).

Interphase extension after cycle 10 has been explained in two ways. Decreasing CycB has been observed to correlate with longer interphase after cycle 10, and it was thus proposed that interphase extension after cycle 10 was due to CycB limitation. However, based on the observation that fast cycles continue after cycle 10 in grp mutant embryos it has been proposed that in wild-type embryos depletion of factors involved in DNA replication causes longer interphase after cycle 10 and the interphase extensions are regulated by the DNA-replication checkpoint pathway (Ji, 2004 and references therein).

Several observations of the current study might resolve this controversy. Interphase extension was observed to occur in grp mutant embryos before cycle 10, thus it is proposed that interphase extension before cycle 10 is solely due to CycB limitation. This is further supported by the following observations: (1) interphase extension occurs at an earlier cycle when maternal CycB is reduced and later when CycB is increased; (2) looking within a specific cycle, interphase is longer when CycB is lower and shorter when CycB is higher; (3) global CycB levels start to oscillate at the beginning of cycle 6 or 7 in wild-type embryos, exactly the same time when interphase duration starts to increase (Ji, 2004).

It is also proposed that after cycle 10, interphase extension is under control of both CycB limitation and the DNA-replication checkpoint. It was reported that grp1 is a null allele. Interphase continuously extends in grp1 embryos after cycle 10, although this extension is not as extensive as in wild-type embryos. This observation supports the idea that limitation of CycB is responsible for this increase (Ji, 2004).

Control of DNA replication and chromosome ploidy by geminin and cyclin A: The geminin deficiency-induced phenotype is partially suppressed by coablation of Chk1/Grapes

Alteration of the control of DNA replication and mitosis is considered to be a major cause of genome instability. To investigate the mechanism that controls DNA replication and genome stability, RNAi was used to eliminate the Drosophila geminin from Schneider D2 (SD2) cells. Silencing of geminin by RNAi in SD2 cells leads to the cessation of mitosis and asynchronous overreplication of the genome, with cells containing single giant nuclei and partial ploidy between 4N and 8N DNA content. The effect of geminin deficiency is completely suppressed by cosilencing of Double parked (Dup), the Drosophila homologue of Cdt1, a replication factor to which geminin binds. The geminin deficiency-induced phenotype is also partially suppressed by coablation of Chk1/Grapes, indicating the involvement of Chk1/Grapes in the checkpoint control in response to overreplication. The silencing of cyclin A, but not of cyclin B, also promotes the formation of a giant nucleus and overreplication. However, in contrast to the effect of geminin knockout, cyclin A deficiency leads to the complete duplication of the genome from 4N to 8N. The silencing of geminin causes rapid downregulation of Cdt1/Dup, which may contribute to the observed partial overreplication in geminin-deficient cells. Analysis of cyclin A and geminin double knockout suggests that the effect of cyclin A deficiency is dominant over that of geminin deficiency for cell cycle arrest and overreplication. Together, these studies indicate that both cyclin A and geminin are required for the suppression of overreplication and for genome stability in Drosophila cells (Mihaylov, 2002).

Human geminin was originally isolated during the analysis of proteins that are associated with human Chk2 protein. While this interaction appeared to be relatively weak during later verification, attempts have been made to address its potential significance for SD2 cells. In SD2 cells, Chk2 knockout did not have a significant effect on geminin deficiency-induced overreplication or the formation of giant nuclei. However, Chk1/Grapes deficiency significantly suppressed the geminin knockout phenotype, suggesting that Chk1/Grapes possesses a checkpoint function for the overreplication induced by geminin deficiency. This result is consistent with those of previous studies indicating that Chk1/Grapes regulates the DNA replication checkpoint for Drosophila. These studies have shown that interference of Drosophila nuclear division cycles 12 and 13 by X-irradiation or the DNA replication inhibitor aphidicolin activates the Chk1/Grapes signaling pathway. It has been shown that the activated Chk1 kinase phosphorylates Cdc25, promoting its complex formation with 14-3-3 and its subsequent retention in cytoplasm. Consequently, the activated Chk1/Grapes promotes the inhibitory phosphorylation of Cdc2 at threonine14 and tyrosine15 in a Cdc25/String-dependent process. In the current studies, it is likely that overreplication caused by geminin deficiency induces the Chk1/Grapes-mediated checkpoint, leading to the inhibition of the Cdc2 kinase activity and mitosis. This effect may be reflected in part by the observation that cyclin B-associated kinase activity is not dramatically induced by geminin deficiency compared to the marked increase of cyclin B protein levels in these cells. Since the loss of Chk1/Grapes or Cdt1 can either partially or completely rescue the geminin deficiency-induced phenotypes, these studies indicate that the loss of Chk1/Grapes does not suppress geminin deficiency through downregulation of Cdt1. Instead, the loss of Chk1/Grapes partially restores the Cdt1 levels in geminin-deficient cells. In mouse cells, Chk1 deficiency causes an aberrant G2/M cell cycle checkpoint during development or in response to DNA damage, causing the formation of nuclei containing highly condensed and aggregated chromatin and, consequently, massive apoptotic cell death. Although no extensive cell death was observed in Chk1/Grapes knockout SD2 cells, it is possible that Chk1/Grapes silencing allows cells to undergo aberrant mitosis, even though they are overreplicating their genome. This would produce a pseudorescue effect on the geminin deficiency-induced phenotypes. It is unclear how Chk1/Grapes rescues Cdt1/Dup expression. It is possible that Chk1/Grapes may be involved in the suppression of Cdt1/Dup transcription during overreplication. Alternatively, since Cdt1/Dup protein levels are regulated in a cell cycle-dependent fashion, being high in G1 and low in S and G2/M phases, the aberrant mitosis and possibly subsequent G1 phase induced by Chk1/Grapes deficiency may allow Cdt1/Dup to be expressed in G1. Further work is required to clarify these issues. Although no significant effect of Chk2 is seen, Chk2 may play a regulatory role for geminin under certain conditions (Mihaylov, 2002).

Genome instability is often associated with cancer. It is still not clear how these processes are linked to the alteration of DNA replication, mitosis, or G1 cell cycle regulation. The present work suggests that dysregulation of geminin/Cdt1 and cyclin A contributes to genome instability in Drosophila cells. Further studies are necessary to link alterations in the activities of geminin/Cdt1 and mitotic cyclins to human cancer (Mihaylov, 2002).

Relative contribution of DNA repair, cell cycle checkpoints, and cell death to survival after DNA damage in Drosophila larvae

Components of the DNA damage checkpoint are essential for surviving exposure to DNA damaging agents. Checkpoint activation leads to cell cycle arrest, DNA repair, and apoptosis in eukaryotes. Cell cycle regulation and DNA repair appear essential for unicellular systems to survive DNA damage. The relative importance of these responses and apoptosis for surviving DNA damage in multicellular organisms remains unclear. After exposure to ionizing radiation, wild-type Drosophila larvae regulate the cell cycle and repair DNA; grp (DmChk1) mutants cannot regulate the cell cycle but repair DNA; okra (DmRAD54) mutants regulate the cell cycle but are deficient in repair of double strand breaks (DSB); mei-41 (DmATR) mutants cannot regulate the cell cycle and are deficient in DSB repair. All undergo radiation-induced apoptosis. p53 mutants regulate the cell cycle but fail to undergo apoptosis. Of these, mutants deficient in DNA repair, mei-41 and okra, show progressive degeneration of imaginal discs and die as pupae, while other genotypes survive to adulthood after irradiation. Survival is accompanied by compensatory growth of imaginal discs via increased nutritional uptake and cell proliferation, presumably to replace dead cells. It is concluded that DNA repair is essential for surviving radiation as expected; surprisingly, cell cycle regulation and p53-dependent cell death are not. It is proposed that processes resembling regeneration of discs act to maintain tissues and ultimately determine survival after irradiation, thus distinguishing requirements between muticellular and unicellular eukaryotes (Jaklevic, 2004).

In eukaryotes, DNA damage checkpoints monitor the state of genomic DNA and delay the progress through the cell cycle as needed. Central components of this checkpoint in mammals include four kinases: ATM, ATR, Chk1, and Chk2. Homologs of these exist in other eukaryotes and assume similar roles where examined. Human patients with ATM mutations, as well as their cells, show a dramatic sensitivity to killing by ionizing radiation. The importance of checkpoints in cellular survival to DNA damaging agents is presumed to be due to the role of checkpoints in cell cycle regulation. This is because mutants in the budding yeast gene rad9, the first checkpoint gene to be characterized, fail to arrest the cell cycle following damage and show increased radiation sensitivity; the latter phenotype is rescued by experimental induction of cell cycle delay. Consequently, cell cycle delay is thought to allow time for DNA repair and thereby ensure survival (Jaklevic, 2004 and references therein).

Components of the DNA damage checkpoint are found to activate DNA repair and to promote programmed cell death, which would cull cells with damaged DNA. For example, phosphorylation of NBS (a component of the Mre11 repair complex) by human ATM is of functional importance, while ATM knockout mice show a reduction in radiation-induced cell death in the CNS. Therefore, the essential role of checkpoints in conferring survival to genotoxins may be due to DNA repair and cell death responses in addition to or instead of cell cycle regulation. Furthermore, what is important for survival at the cellular level may not be so in a multicellular context. For instance, the failure to arrest the cell cycle by checkpoints may be detrimental to individual cells, but removal of these by cell death and replacement via organ homeostasis may make cell cycle regulation inconsequential for survival of multicellular organs (Jaklevic, 2004).

To address how DNA damage checkpoints operate in the context of multicellular organisms in vivo, the effect of ionizing radiation on Drosophila melanogaster is being studied. In Drosophila, mei-41 (ATR homolog) and grp (Chk1 homolog) are required to delay the entry into mitosis in larval imaginal discs after irradiation and to delay the entry into mitosis after incomplete DNA replication in the embryo. Thus, mei-41 and grp play similar roles to their homologs in other systems. Moreover, mei-41 mutants are deficient in DNA repair. The role of mei-41 and grp in radiation-induced cell death has not been tested, but mei-41 is dispensable for cell death after enzymatic induction of DNA double-strand breaks (Jaklevic, 2004 and references therein).

Mutants in mei-41, grp, p53, and okra, a homolog of budding yeast RAD54 that functions in repair of DNA double-strand breaks (DSB) have been used to address the relative importance of cell cycle regulation, cell death, and DNA repair to the ability of a multicellular organism to survive ionizing radiation. The three responses are affected to different degrees in these mutants: wild-type larvae regulate S and M phases and repair DNA; grp mutants are unable to regulate the cell cycle but are able to repair DNA; okra mutants are able to regulate the cell cycle but are deficient in DNA repair; and mei-41 mutants are unable to regulate the cell cycle and are also deficient in DNA repair. All genotypes with the exception of p53 mutants are proficient in radiation-induced cell death, suggesting that mei-41 and grp do not contribute to this response. Under these conditions, it is found that while mei-41 and okra mutants are highly sensitive to killing by ionizing radiation, p53 mutants show reduced but significant survival and grp mutants resemble wild-type. These results suggest that cell death is neither sufficient nor absolutely necessary, DNA repair is essential, and optimal cell cycle regulation is dispensable for surviving ionizing radiation in Drosophila larvae (Jaklevic, 2004).

The effects of DNA damage by ionizing radiation on the maintenance and survival of Drosophila larvae was studied. Despite an extensive loss of cells to radiation-induced cell death, organ size and morphology are maintained remarkably well, and larvae survive to produce viable adults. Surprisingly, optimal cell cycle regulation by checkpoints is neither necessary (as in grp mutants) nor sufficient (as in okra mutants) to ensure organ homeostasis and organismal survival. p53-dependent cell death is also largely dispensable in this regard. Instead, DNA repair appears to be of paramount importance as might be expected (Jaklevic, 2004).

In mitotically proliferating cells of Drosophila larval imaginal discs and brains, the first responses to sublethal doses of irradiation (1000R-4000R) are delays in cell cycle progression at 1-2 hr after irradiation, followed by the induction of cell death at 4 hr after irradiation. DNA synthesis resumes at 5 hr after irradiation, while mitotic index resumes at 6 hr after. These relatively early responses are followed by an increase in proliferation that is detectable about a day after irradiation. Presumably, abundant cell death removes damaged cells, but sustained proliferation compensates to maintain proper organ size and morphology. Continued cell proliferation, it is proposed, delays the onset of the next major developmental transition, pupariation. The extent of delay correlates with radiation dose, presumably because more cells are lost at higher doses, requiring more compensatory proliferation (Jaklevic, 2004).

Another response monitored was DNA repair, a substantial portion of which must occur within 3 hr after 220R of irradiation because a significant difference is seen in the incidence of chromosome breakage between wild-type and repair-deficient mutants by this time. However, cytologically visible chromosome breaks likely represent only a fraction of total DNA damage; for this reason, it is not certain if DNA repair is complete within this time frame (Jaklevic, 2004).

Having determined the sequence of responses to irradiation in wild-type larvae, deviations from it in various mutants was documented. mei-41 and grp mutants are unable to dampen DNA synthesis after irradiation. Previous work has shown that both mutants are unable to inhibit mitosis after irradiation, although grp mutants appear to retain a partial activity in this regard. Thus, Drosophila ATR and Chk1 are needed for optimal regulation of both S and M phases after exposure to ionizing radiation. However, induction of cell death does not require mei-41 or grp. The most striking result report in this study is that grp mutants that are defective in regulation of both S and M phases are not sensitive to killing by 2000R of X-rays, doses that readily killed mei-41 and okra mutants. This finding strongly suggests that cell cycle regulation by checkpoints is not absolutely necessary for surviving irradiation under these conditions (Jaklevic, 2004).

In determining what is necessary, the phenotype of okra mutants that can regulate both S and M phases and promote cell death is particularly informative because they are radiation sensitive. Thus, DNA repair is essential, suggesting that it is this defect in mei-41 mutants that renders them radiation sensitive. It is speculated that irradiated mei-41 and okra larvae may attempt to increase proliferation, but the continual presence of unrepaired DNA likely channels these cells to death. This would lead to an eventual decline in cell number, which would undermine maintenance of cellular differentiation that is the basis of the eye disc's morphogenetic furrow (MF). Signals from cells in the MF are thought to be important for the generation of the second mitotic wave. Loss of the MF could then explain the absence of the expected pattern of mitoses in mei-41 and okra discs (Jaklevic, 2004).

Traditionally, checkpoints refer to the regulation of the cell cycle. Recent views propose the inclusion of the other responses among checkpoint responses, such as the preservation of DNA replication intermediates, transcriptional activation, and DNA repair. The data suggest that other responses may be more important in ensuring survival of multicellular organs and organisms. Interestingly, results from budding yeast also question the idea that cell cycle regulation by checkpoints is essential for surviving genotoxins even at the cellular level. For example, yeast Chk1 mutants show profoundly defective regulation of mitosis after irradiation and yet are only mildly radiation sensitive. Another recent study indicates that stabilization of replication forks is crucial for surviving the alkylating agent MMS whereas the ability to inhibit mitosis is less important (Jaklevic, 2004).

It is emphasized that survival in this study refers to that of organs and organisms. At the cellular level, cell cycle regulation by checkpoints may well be crucial to allow time for DNA repair and for survival. In grp mutants that are defective for cell cycle checkpoints but are proficient for DNA repair, cells that progressed through S and M phases with damaged DNA may have been subject to cell death. Indeed, incidence of cell death appears higher in grp (and mei-41) mutants than in wild-type. Loss of these cells, however, is clearly of little consequence to survival of imaginal discs and larvae. This could be because grp mutants are able to repair DNA in cells that are not in S and M phases, i.e., those in G1 or G2. These cells may then proliferate to compensate for lost cells. Numerous studies on tissue regeneration demonstrate the power of Drosophila larvae to restore not only cell number but also proper differentiation. In such a system, the failure of cell cycle checkpoints after irradiation may be of little consequence as long as damaged cells are replaced. It is speculated that these findings may be particularly applicable to multicellular systems with similar regenerative powers such as the human liver (Jaklevic, 2004).

grp (chk1) replication-checkpoint mutations and DNA damage trigger a Chk2-dependent block at the Drosophila midblastula transition

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).

Drosophila ATR in double-strand break repair

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).

Unprotected Drosophila melanogaster telomeres activate the spindle assembly checkpoint

In both yeast and mammals, uncapped telomeres activate the DNA damage response (DDR) and undergo end-to-end fusion. Previous work has shown that the Drosophila HOAP protein, encoded by the caravaggio (cav) gene, is required to prevent telomeric fusions. This study shows that HOAP-depleted telomeres activate both the DDR and the spindle assembly checkpoint (SAC). The cell cycle arrest elicited by the DDR was alleviated by mutations in mei-41 (encoding ATR), mus304 (ATRIP), grp (Chk1) and rad50 but not by mutations in tefu (ATM). The SAC was partially overridden by mutations in zw10 (also known as mit(1)15) and bubR1, and also by mutations in mei-41, mus304, rad50, grp and tefu. As expected from SAC activation, the SAC proteins Zw10, Zwilch, BubR1 and Cenp-meta (Cenp-E) accumulated at the kinetochores of cav mutant cells. Notably, BubR1 also accumulated at cav mutant telomeres in a mei-41-, mus304-, rad50-, grp- and tefu-dependent manner. These results collectively suggest that recruitment of BubR1 by dysfunctional telomeres inhibits Cdc20-APC function, preventing the metaphase-to-anaphase transition (Musarò, 2008).

In most organisms, telomeres contain arrays of tandem G-rich repeats added to the chromosome ends by telomerase. Drosophila telomeres are not maintained by the activity of telomerase, but instead by the transposition of three specialized retrotransposons to the chromosome ends. In addition, whereas yeast and mammalian telomeres contain proteins that recognize telomere-specific sequences, Drosophila telomeres are epigenetically determined, sequence-independent structures. Nonetheless, Drosophila telomeres are protected from fusion events, just as their yeast and mammalian counterparts are. Genetic and molecular analyses have thus far identified eight loci that are required to prevent end-to-end fusion in Drosophila: effete (eff, also known as UbcD1), which encodes a highly conserved E2 enzyme that mediates protein ubiquitination; Su(var)205 and caravaggio (cav), encoding HP1 and HOAP, respectively; the Drosophila homologs of the ATM, RAD50, MRE11A and NBN (also known as NBS1) genes; and without children (woc), whose product is a putative transcription factor (Musarò, 2008).

To determine whether mutations in genes required for telomere capping also affect cell cycle progression, DAPI-stained preparations of larval brains from seven of these eight telomere-fusion mutants were examined. Mutant brains were examined for the mitotic index (MI) and the frequency of anaphases (AF). The mitotic indices observed for the eff, Su(var)205, mre11, rad50, woc and tefu mutants ranged from 0.46 to 0.75, values that were slightly lower than the mitotic index observed for the wild type (0.86). However, brains from cav mutants showed a fourfold reduction of the mitotic index (0.19) with respect to the wild type. cav mutants also had a very low frequency of anaphases (1.7%-1.9%) compared to the wild type (13.2%), whereas in the other mutants, frequency of anaphases ranged from 8.6% to 12.5%. Reductions in both the mitotic index and the frequency of anaphases were rescued by a cav+ transgene, indicating that these phenotypes were indeed due to a mutation in cav (Musarò, 2008).

These results prompted a focus on cav mutations in order to determine how unprotected telomeres might influence cell cycle progression. The cav allele used in this study is genetically null for the telomere-fusion phenotype. cav homozygotes and cav1/Df(3R)crb-F89-4 hemizygotes show very similar mitotic indices and frequencies of anaphases, indicating that cav is also null for these cell cycle parameters. The cav-encoded HOAP protein localizes exclusively to telomeres; cav produces a truncated form of HOAP that fails to accumulate at chromosome ends (Musarò, 2008).

The low frequencies of anaphases observed in cav mutant cells suggest that they may be arrested in metaphase. To confirm a metaphase-to-anaphase block, mitoses were filmed of cav and wild-type neuroblasts expressing the GFP-tagged H2Av histone. Control cells entered anaphase within a few minutes after chromosome alignment in metaphase, whereas cav cells remained arrested in metaphase for the duration of imaging (Musarò, 2008).

It was hypothesized that the cav-induced metaphase arrest was the result of SAC activation. As in all higher eukaryotes, unattached Drosophila kinetochores recruit three SAC protein complexes (Mad1-Mad2, Bub1-BubR1-Cenp-meta and Rod-Zw10-Zwilch) that prevent precocious sister chromatid separation by negatively regulating the ability of Cdc20 to activate the anaphase-promoting complex or cyclosome (APC/C). Mutations in genes encoding components of these complexes lead to SAC inactivation and allow cells to enter anaphase even if the checkpoint is not satisfied. To ask whether the low frequency of anaphases in cav mutant brains was due to SAC activation, zw10 cav and bubR1 cav double mutants were analyzed. In both cases, the frequency of anaphases was significantly higher than in the cav single mutant, whereas the frequency of telomere fusions remained unchanged. These results imply that the low frequency of anaphases in cav mutants is indeed due to SAC activation (Musarò, 2008).

SAC activation would be expected to increase the mitotic index through the accumulation of metaphase cells; however, in cav single mutants, the mitotic index is abnormally low. One explanation for this apparent paradox is that the cell cycle in cav cells is also delayed before M-phase, as a result of the DNA damage response (DDR). To ask whether HOAP-depleted telomeres activate any DNA damage checkpoints, double mutants were generated for cav and genes known to be involved in these checkpoints: mei-41 and telomere fusion (tefu), encoding the fly homologs of ATR and ATM, respectively; mus304, which encodes the ATR-interacting protein ATRIP grapes (grp), which specifies a CHK1 homolog and rad50, whose product is part of the Mre11-Rad50-Nbs complex. DAPI-stained preparations of larval brain cells from these double mutants showed that mei-41, mus304, grp and rad50 mutations alleviate the cell cycle block induced by cav, causing a ~2.5-fold increase of the mitotic index relative to that observed in the cav single mutant. In contrast, the tefu mutation did not affect the cav- induced interphase block. These effects are unrelated to variations in the frequency of telomere fusions, as the telomere fusion frequencies in double mutants were very similar to those in the cav single mutant. It is thus concluded that the interphase arrest in cav mutants occurs independently of ATM and is mediated by a signaling pathway involving ATR, ATRIP, Chk1 and Rad50. This signaling pathway is known to activate DNA damage checkpoints during the G1/S transition, the S phase and the G2/M transition. However, the current results do not allow identification of the particular checkpoint(s) activated by HOAP-depleted telomeres (Musarò, 2008).

Notably, in all double mutants for cav and any one of the genes associated with the DDR, including tefu (ATM), a significant increase was also observed in the frequencies of anaphases relative to that of the cav single mutant, suggesting that these genes are involved in the cav-induced metaphase arrest. This finding reflects a role of these DDR-associated genes in the peculiar mechanism by which uncapped Drosophila telomeres activate SAC (Musarò, 2008).

To obtain further insight about the cav-induced metaphase arrest, the localization of Zw10, Zwilch, BubR1 and Cenp-meta (Cenp-E) was determined by immunofluorescence. In wild-type Drosophila cells, these proteins begin to accumulate at kinetochores during late prophase and remain associated with kinetochores until the chromosomes are stably aligned at the metaphase plate. Treatments with spindle poisons (for example, colchicine) disrupt microtubule attachment to the kinetochores, leading to metaphase arrest with SAC proteins accumulated at the centromeres. Immunostaining for Zwilch, Zw10, Cenp-meta or BubR1 showed that in all cases, the frequencies of cav metaphases with strong centromeric signals were comparable to those observed in colchicine-treated wild-type cells, and they were significantly higher than those seen in untreated wild-type metaphases. These findings support the view that HOAP-depleted telomeres activate the canonical SAC pathway (Musarò, 2008).

Through a detailed examination of cav metaphases immunostained for SAC proteins, an unexpected connection was found between uncapped telomeres and the localization of at least one SAC component. Although Zwilch, Zw10 and Cenp-meta accumulated exclusively at kinetochores, BubR1 was concentrated at both kinetochores and telomeres. BubR1 localized at both unfused (free) and fused telomeres; most (94.4%) cav metaphases showed at least one telomeric BubR1 signal. To better resolve the chromosome tangles seen in cav metaphases, cells were treated with hypotonic solution, allowing a focus on free telomeres, which can be reliably scored. It was found that 25% of the free telomeres in cav metaphases show an unambiguous BubR1 signal. BubR1 accumulations were not observed at wild-type telomeres or at the breakpoints of X-ray-induced chromosome breaks. BubR1 localization at telomeres was not caused by the formation of ectopic kinetochores at the chromosome ends, since cav telomeres did not recruit the centromere and kinetochore marker Cenp-C. Low frequencies of BubR1-labeled telomeres were also observed in other mutant strains with telomere fusions including eff, Su(var)205 and woc. These results indicate that BubR1 specifically localizes at uncapped telomeres (Musarò, 2008).

It was next asked whether mutations in mei-41, grp, mus304, tefu, rad50 and zw10 affect BubR1 localization at cav mutant telomeres. Whereas mutations in zw10 did not affect BubR1 localization at cav chromosome ends, double mutants for cav and any of the other genes all showed significant reductions in the frequency of BubR1-labeled free telomeres with respect to cav single mutants. Considered together, these results indicate that when the canonical SAC machinery is intact (in all cases except in zw10 cav double mutants), there is a strong negative correlation between the frequency of BubR1-labeled telomeres and the frequency of anaphases. These findings suggest that BubR1 accumulation at telomeres can activate the SAC (Musarò, 2008).

Finally it was asked whether mutations in DDR-associated genes can allow cells to bypass the SAC when it is activated by spindle abnormalities rather than by uncapped telomeres. The spindle was disrupted in two ways: with the microtubule poison colchicine and with mutations in abnormal spindle (asp). Both situations activated the SAC and caused metaphase arrest; neither mei-41 nor grp or tefu mutations allowed cells to bypass this arrest, whereas mutations in zw10 led such cells to exit mitosis. These findings indicate that the DDR-associated genes regulate BubR1 accumulation at cav telomeres but are not directly involved in the SAC machinery (Musarò, 2008).

Collectively, these results suggest a model for the activation of cell cycle checkpoints by unprotected Drosophila telomeres. It is proposed that uncapped telomeres activate DDR checkpoints, leading to interphase arrest through a signaling pathway involving mei-41 (encoding ATR), mus304 (ATRIP), grp (Chk1) and rad50, but not tefu (ATM). This pathway is independent of telomeric BubR1, because mutations in tefu, which strongly reduce BubR1 accumulation at chromosome ends, do not rescue cav-induced interphase arrest. Uncapped telomeres can also activate the SAC by recruiting BubR1 through a pathway requiring mei-41, mus304, grp, rad50 and tefu functions. Once accumulated at the telomeres, BubR1 may negatively regulate either Fizzy (Cdc20) or another APC/C subunit so as to cause metaphase arrest. This model posits that certain DDR-associated genes, such as rad50, function both in the DDR pathway and in the pathway that mediates BubR1 recruitment at telomeres. This explains why rad50 and mre11 mutants show only mild reductions of the mitotic index and the frequency of anaphases even though HOAP is substantially depleted from their telomeres (Musarò, 2008).

It is proposed that uncapped telomeres can induce an interphase arrest independently of BubR1 through a signaling pathway that involves ATR, ATRIP, CHK1 and Rad50 but not ATM. The same proteins, including ATM, are required for the recruitment of BubR1 at unprotected telomeres. Telomeric BubR1 may negatively regulate the activity of the Cdc20-APC complex, leading to a metaphase-to-anaphase transition block. The metaphase arrest caused by Cdc20-APC inhibition is likely to cause an accumulation of SAC proteins on the kinetochores, reinforcing SAC activity. Consistent with this view, mutations in ida, which encodes an APC/C subunit, lead to a metaphase arrest phenotype with BubR1 accumulated at the kinetochores (Musarò, 2008).

Several recent reports have suggested possible relationships between DNA damage, SAC and telomeres. In both Drosophila and mammalian cells, DNA breaks can activate the SAC, presumably by disrupting kinetochore function. In Schizosaccharomyces pombe, Taz1-depleted telomeres result in Mph1p- and Bub1p-mediated SAC activation, and mutations in yKu70 affecting Saccharomyces cerevisiae telomere structure also activate the SAC. However, these previous studies did not explain how telomere perturbations might be perceived by the SAC. This study has found that unprotected Drosophila telomeres recruit the BubR1 kinase as do the kinetochores that are unconnected to spindle microtubules. Thus, it is possible that telomere-associated BubR1 inhibits anaphase through molecular mechanisms similar to those that govern SAC function at the kinetochore. Consistent with this possibility, a single BubR1 accumulation at either a centromere or a telomere seems competent to block anaphase onset. It will be of interest in the future to establish whether deprotected mammalian telomeres can also activate the SAC and, if so, whether BubR1 recruitment to the damaged telomeres mediates this response (Musarò, 2008).

Genetic screen in Drosophila melanogaster uncovers a novel set of genes required for embryonic epithelial repair

The wound healing response is an essential mechanism to maintain the integrity of epithelia and protect all organisms from the surrounding milieu. In the 'purse-string' mechanism of wound closure, an injured epithelial sheet cinches its hole closed via an intercellular contractile actomyosin cable. This process is conserved across species and utilized by both embryonic as well as adult tissues, but remains poorly understood at the cellular level. In an effort to identify new players involved in purse-string wound closure a wounding strategy suitable for screening large numbers of Drosophila embryos was developed. Using this methodology, wound healing defects were observed in Jun-related antigen (encoding DJUN) and scab (encoding Drosophila alphaPS3 integrin) mutants and a forward genetics screen was performed on the basis of insertional mutagenesis by transposons that led to the identification of 30 lethal insertional mutants with defects in embryonic epithelia repair. One of the mutants identified is an insertion in the karst locus, which encodes Drosophila betaHeavy-spectrin. betaHeavy-spectrin (betaH) localizes to the wound edges where it presumably exerts an essential function to bring the wound to normal closure (Campos, 2010).

Using previously described DC or wound healing mutants a pilot screen was performed to validate the embryonic wounding strategy. The fact that a member of the DJNK pathway (Jra/DJun) was identified in the assay is in accordance with other reports that implicate this pathway in wound healing. Specifically, two mutations in components of the DJNK pathway, bsk/DJNK and kay/DFos, were previously shown to have defects in fly larval and adult wound closure, respectively. In addition, a reporter construct has been describes that requires consensus binding sites for the JUN/FOS complex to be activated upon wounding. Interestingly, treporter activation was still observed in Jra mutants, which suggests that additional signaling pathways are involved in wound closure (Campos, 2010).

An apparent discrepancy arose when the assay revealed a phenotype with Jra but not with puc mutants, another component of the same signaling pathway. This result might be explained by the fact that Jra and puc function in opposite directions in the DJNK signaling pathway. Puc functions as a pathway repressor, so in a puc mutant the JNK pathway should be less repressed and an opposite effect to a Jra mutation could be expected. In addition, activation of a puc-lacZ reporter has been shown to occur in larvae, wing imaginal discs, and adult wounds that take 18-24 hr to close, but it is only robustly detectable 4-6 hr postpuncture. Embryonic wounds are faster to heal, and even after inflicting a large laser wound on stage 14/15 embryos, no activation of the puc-lacZ reporter (assessed in open wounds 3 hr postwounding by immunofluorescence; data not shown) was detected. This observation suggests that, in rapidly healing epithelial wounds, the JNK pathway is not activated to high enough levels to trigger auto-inhibition (Campos, 2010).

The α-integrin scab was never before implicated in embryonic wound healing, but this mutant's phenotype comes as no great surprise. The first scab mutation was isolated due to its abnormal larval cuticle patterning. The scab gene encodes for Drosophila α-PS3 integrin, which is zygotically expressed in embryonic tissues undergoing invagination, tissue movement, and morphogenesis. Integrin proteins are involved in cell-matrix interactions and α-PS3 integrin regulation, in particular, mediates zipping of opposing epithelial sheets during DC. Similarly, the observation of a wound defect in scb5J38 mutants is consistent with a role for α-PS3 integrin in zipping of opposing epithelial cells during the healing process (Campos, 2010).

A previous study using confocal video microscopy has shown that Rho11B mutants take twice as long to close an epithelial wound when compared to wild type. Rho1 was confirmed in the assay to be important for wound healing, although with a weaker phenotype (22% of embryos had unclosed holes). This result shows nonetheless that the assay can be sensitive enough to pick up a 'weak' wound healing mutant such as Rho11B, which is still able to heal wounds albeit slower than wild type (Campos, 2010).

The genes identified in the screen represent a variety of functions indicating that wound healing is a complex mechanism that requires the participation of many cellular processes. A large class of the candidate mutants are involved in several aspects of gene expression, including factors that regulate chromatin remodeling (dUtx and Pc), elongation (dEaf), splicing (Glo and CG3294), and translation (CG33123). These factors are likely needed during wound healing for the induction of a repair transcriptome. Interestingly, JNK signaling-dependent Pc group (PcG) gene downregulation has been observed during imaginal disc regeneration. In addition, a recent study revealed that PcG methylases are downregulated during wound healing, while counteracting demethylases, Utx and Jmjd3, are upregulated. The results for the Pc and Utx mutants are consistent with these studies and highlight the importance of epigenetic reprogramming in the repair process (Campos, 2010).

Some of the genes such as arc-p20 and karst probably have a more direct role in the cell shape changes that drive the tissue morphogenetic movements during epithelial repair. The gene product of arc-p20 is a component of Arp2/3, a complex that controls the formation of actin filaments, and karst encodes a component of the spectrin membrane cytoskeleton. Also related to morphogenesis, CG12913 encodes an enzyme involved in the synthesis of chondroitin sulfate, which is usually found attached to proteins as part of a proteoglycan, suggesting a predictable contribution of the extracellular matrix in the tissue movements necessary for wound healing (Campos, 2010).

The epithelium is the first line of defense of the organism against pathogens and tissue integrity. It would thus seem plausible that genes involved in innate immunity could be identified with the screening protocol. Indeed, two of the genes (Ser12 and CG5198) seem to point to the involvement of the immune response in the healing of the laser-induced wounds. Ser12 is a member of the serine protease family, a class of proteins that has been shown to play a role in innate immunity. The CG5198 gene has no described function in Drosophila so far, but its homolog, CD2-binding protein 2, is involved in T lymphocyte activation and pre-RNA splicing. Another candidate that might represent a link to immunity is Atg2, a gene important for the regulation of autophagy, a process by which cells degrade cytoplasmic components in response to starvation. In Drosophila, autophagy has been linked to the control of cell growth, cell death, and, recently, to the innate immune response mechanism against vesicular stomatitis virus and listeria infection (Campos, 2010).

Isolation of an insertion in the stam gene points to the involvement of the JAK-STAT signaling cascade in this regenerative process. Interestingly, stam has been shown to be involved in Drosophila tracheal cell migration and is upregulated following Drosophila larvae infection by Pseudomonas entomophila (Campos, 2010).

One candidate could be involved in the uptake or export of some important wound signal (CG7627) as this gene encodes for a multidrug resistant protein (MRP), part of the ABC transporter superfamily, involved in drug exclusion properties of the Drosophila blood-brain barrier (Campos, 2010).

The kinase encoded by grapes is the Drosophila homolog of human Check1 (Chk1) involved in the DNA damage and mitotic spindle checkpoints. All the Chk1 literature has focused on its role during the cell cycle. However, the Drosophila late embryonic epithelium is a quiescent tissue, even after wounding. Understanding Grapes function in this context is a challenging task that could lead to new paradigms. One hypothesis is that Grapes is involved in tension sensing, as it is in the spindle checkpoint, or may uncover a cellular repair process that could help damaged cells 'decide' to either die by apoptosis or participate in the repair process (Campos, 2010).

The remaining genes with a putative function represent a wide range of general metabolic processes (aralar1, gs1-like, CG4389, CG9249, CG11089, and CG16833), suggesting that healing the epithelium is a highly demanding process (Campos, 2010).

Finally, a significant number of genes that have not yet been studied and do not contain identifiable protein domains (CG2813, CG31805, CG6005, CG6750, CG10217, CG15170, and CG30010) were selected. At the moment it is not possible to predict the role that these genes may play, but further study may help to identify novel wound healing regulatory mechanisms (Campos, 2010).

One of the mutants identified in the transposon screen was kstd11183, an insertion in the βH-spectrin locus. This mutation is likely producing a truncated protein terminating three amino acids into the P-element insertion. Other mutations identified in nearby segments 14 (kst14.1, kst2) and 16 (kst1) lead to the production of a detectable truncated protein so it is likely that karstd11183 mutation also gives rise to a truncated protein. These mutant forms of βH lack approximately half of the wild-type protein, including a COOH-terminal PH domain region, which is involved in targeting the protein to the membrane, thus producing a potential dominant negative form of βH. However, the karstd11183 mutant should still have maternally loaded wild-type protein, as previous studies describe a complete absence of maternal protein only by the third instar larval stage. This maternal contribution is likely the main reason that this mutant, as well as the other mutants isolated in the screen, does not have a fully penetrant wound healing phenotype (Campos, 2010).

βH-spectrin was shown to localize to the actomyosin purse string, a supracellular contractile cable that forms rapidly upon wound induction. Live imaging has demonstrated that actin and myosin can accumulate in this cable structure within minutes after wounding. Unfortunately, due to the size of the βH gene (>13 kb) cloning and tagging it for live imaging is not possible using standard methods, but the experiments in fixed tissue reveal that βH can accumulate very rapidly in this cable structure. βH accumulation was observed at the earliest time point technically feasible, 15 min postwounding. These observations are consistent with previous studies, also in fixed tissue, demonstrating rapid changes in βH localization during the process of cellularization in Drosophila embryos. Taken together, it is clear that at least the βH component of the membrane skeleton is not just a static structural scaffold as the name implies, but rather a dynamic protein capable of responding to or directing changes in cellular dynamics. The studies suggest that polarized redistribution of βH exerts an essential function to facilitate actin-based cellular responses, such as cable accumulation/maintenance and wound edge filopodia dynamics, which are necessary to properly close a wound (Campos, 2010).

βH has been previously observed in association with actin 'rings' during development of Drosophila and C. elegans. Arguably, C. elegans provides an example of actin ring function most analogous to the Drosophila wound edge purse string. During the final stages of C. elegans development, cortical arrays of actin in the outer epithelial cells, the hypodermis, dramatically reorganize to form parallel apically localized bundles of circumferencial supracellular actin rings. In this system, sma1, the C. elegans ortholog of βH, also localizes apically to these actin rings. In sma1 mutants the rings fail to productively contract and begin to disorganize, losing connection to the cell membranes. An additional phenotype observed in these mutants is the inability of cells to change their shape, a process normally 'directed' by these contractile rings, the end result being a short worm, a phenotype seen as functionally analogous to an unclosed wound in the Drosophila system (Campos, 2010).

In Drosophila, βH has been implicated in modulating cell shape changes during apical constriction of follicle cells (a process also involving actin rings) and has been proposed to function as a link between cross-linked actin networks/rings and the cell membrane. Further studies revealed that the C-terminal domain of βH has the ability to directly modulate the apical membrane area by regulating endocytosis, adding one more tantalizing piece of evidence pointing to the fact that βH could be a major player in cell shape changes, not only as a structural link but also by directly modulating the membrane area in response to cytoskeletal clues (or vice versa) (Campos, 2010).

Although it is known from previous studies that the actin cable is not absolutely required for wound closure, the process takes much longer without one. In Rho1 mutant embryos, cells lacking a cable are able to pull the wound closed using filopodia. The filopodial defect observed in karst mutants, adds another line of evidence to the absolute requirement of these structures for wound closure. In addition to the reduced actin cable accumulation and filopodial dynamics in karst mutants (which would lack the C-terminal domain responsible for membrane modulation), a lack of cell shape change is seen in the wound edge cells. Taken together, these data and the published work, introduce the intriguing possibility that βH could be serving as a link between wound edge dynamics and the coordinated cell shape changes usually observed in wild-type wound edge cells. The combination of the proposed ability of βH to modulate the apical membrane area as well as cross-link actin and act as an apical membranewide scaffold for other interactions, makes βH a good candidate to provide the physical link that would coordinate tissuewide actions, such as supracellular actin cable contraction, with the individual cellular responses, such as cell shape change and polarized filopodia activity (Campos, 2010).

Drosophila Claspin is required for the G2 arrest that is induced by DNA replication stress but not by DNA double-strand breaks

ATR and Chk1 are protein kinases that perform major roles in the DNA replication checkpoint that delays entry into mitosis in response to DNA replication stress by hydroxyurea (HU) treatment. They are also activated by ionizing radiation (IR) that induces DNA double-strand breaks. Studies in human tissue culture and Xenopus egg extracts identified Claspin as a mediator that increased the activity of ATR toward Chk1. Because the in vivo functions of Claspin are not known, Drosophila lines were generated that each contained a mutated Claspin gene. Similar to the Drosophila mei-41/ATR and grp/Chk1 mutants, embryos of the Claspin mutant showed defects in checkpoint activation, which normally occurs in early embryogenesis in response to incomplete DNA replication. Additionally, Claspin mutant larvae were defective in G2 arrest after HU treatment; however, the defects were less severe than those of the mei-41/ATR and grp/Chk1 mutants. In contrast, IR-induced G2 arrest, which was severely defective in mei-41/ATR and grp/Chk1 mutants, occurred normally in the Claspin mutant. It was also found that Claspin is phosphorylated in response to HU and IR treatment and a hyperphosphorylated form of Claspin is generated only after HU treatment in mei-41/ATR-dependent and tefu/ATM-independent way. In summary, these data suggest that Drosophila Claspin is required for the G2 arrest that is induced by DNA replication stress but not by DNA double-strand breaks, and this difference is probably due to distinct phosphorylation statuses (Lee, 2012).

Claspin was originally identified in Xenopus laevis egg extracts as a Chk1-interacting protein that was required for DNA replication stress-induced G2 arrest. DNA replication stress induces ATR-dependent phosphorylation of Claspin, which results in a Claspin-Chk1 interaction and phosphorylation and activation of Chk1 by ATR. Claspin protein levels are regulated during the cell cycle, peak at the S/G2 boundary, and are degraded during mitosis. In support of its high expression during S phase, Claspin is reported to have a role during normal DNA replication when in the absence of exogenous DNA damage. Moreover, Claspin is required for terminating DNA damage-induced cell cycle arrest. Phosphorylation of Claspin by Polo-like kinase-1 (Plk1) results in the dissociation of Claspin from chromatin in Xenopus or the degradation of Claspin in human cells, which leads to inactivation of Chk1 and resumption of the cell cycle after prolonged interphase arrest. Most of the work on Claspin has been performed in Xenopus egg extracts and human tissue culture, however, animal models for Claspin have not been reported (Lee, 2012).

To understand the in vivo function of Claspin, mutants of Claspin (CG32251) were generated by imprecise excision of a transposable element. Analysis of the DNA damage checkpoint during early embryogenesis and the larval stage of this mutant showed that Drosophila Claspin is required for cell cycle arrest in response to incompletely replicated DNA. However, Claspin is dispensable for IR-induced cell cycle arrest. Interestingly, Claspin is phosphorylated after IR and HU treatment and a hyperphosphorylated form of Claspin was observed after HU but not after IR treatment. Moreover, the HU-induced hyperphosphorylation of Claspin is attenuated in mei-41/ATR mutant, but not in tefu/ATM mutant. These results suggest that the phosphorylation state and the role of Drosophila Claspin in cell cycle arrest are distinctly regulated by different types of DNA damage: DNA replication stress and DSBs (Lee, 2012).

Different cyclin types collaborate to reverse the S-phase checkpoint and permit prompt mitosis

Precise timing coordinates cell proliferation with embryonic morphogenesis. As Drosophila embryos approach cell cycle 14 and the midblastula transition, rapid embryonic cell cycles slow because S phase lengthens, which delays mitosis via the S-phase checkpoint. This study probed the contributions of each of the three mitotic cyclins to this timing of interphase duration. Each pairwise RNA interference knockdown of two cyclins lengthened interphase 13 by introducing a G2 phase of a distinct duration. In contrast, pairwise cyclin knockdowns failed to introduce a G2 in embryos that lacked an S-phase checkpoint. Thus, the single remaining cyclin is sufficient to induce early mitotic entry, but reversal of the S-phase checkpoint is compromised by pairwise cyclin knockdown. Manipulating cyclin levels revealed that the diversity of cyclin types rather than cyclin level influenced checkpoint reversal. It is concluded that different cyclin types have distinct abilities to reverse the checkpoint but that they collaborate to do so rapidly (Yuan, 2012).

Pairwise knockdown of cyclins in embryos lacking Chk1/Grapes (embryos from grp mutant mothers) modestly extends interphase in the grp embryo. This knockdown also substantially suppresses the mitosis 13 defects in grp embryos. The mitotic catastrophe in grp embryos has long been thought to be caused by entry into mitosis with incompletely replicated DNA. Indeed, analysis of PCNA localization supports a proposal that the small extension of interphase allows completion of S phase and, hence, suppression of the catastrophe. This suppression of the grp phenotype, which extends to partial restoration of gastrulation, is reminiscent of suppression of hypomorphic mei-41 mutations when the maternal dose of CycA and/or CycB was reduced. However, the most important feature of this analysis is that the extension of interphase by pairwise cyclin knockdown in grp embryos is so slight that interphase remains shorter than a wild-type interphase 13. Thus, each individual cyclin type can drive rapid advance to mitosis in the absence of functional Chk1. Furthermore, the G2 that was introduced by cyclin knockdown was absent in grp embryos, and cyclin type-specific differences in interphase length were minor. These results demonstrate that mitotic entry is timed primarily by the Grapes-dependent checkpoint in cyclin knockdown embryos and, moreover, that the G2 induced in these embryos results from action of the checkpoint (Yuan, 2012).

How might S phase govern the time of mitosis when mitosis begins well after completion of S phase? To test whether the S-phase checkpoint delayed accumulation of the remaining cyclin, we immunoblotted single knockdown embryos to follow accumulation in wild-type and grp embryos. Cyclin accumulated during S phase in both wild-type and grp mutant embryo. Thus, Grapes function does not delay the production of cyclin. Instead, the difference between grp+ and grp embryos suggests that persistent activity of the checkpoint prevents the checkpoint-competent embryos from going into mitosis after pairwise cyclin knockdown. Because wild-type embryos enter mitosis immediately after S phase, the checkpoint is rapidly reversed when there is a full complement of cyclin types, but its reversal is delayed upon pairwise cyclin knockdown. Apparently, the different cyclin types ordinarily collaborate to rapidly reverse the checkpoint (Yuan, 2012).

This study shows that a persistent action or consequence of the DNA replication checkpoint underlies a G2 phase that is introduced by pairwise knockdown of cyclins. One might propose that cyclin knockdown doesn't really cause persistence of the checkpoint activity but simply makes a slowly decaying checkpoint function longer by compromising the cyclin-Cdk1 activity that must be suppressed by the checkpoint. Such an interpretation is disfavored because it is quantitative, and the data argue that neither reduction nor increase in the remaining cyclin affects the duration of interphase. Instead, it is argued that cyclin-Cdk1 contributes to shutting off the checkpoint, and it is proposed that efficient shutoff of the checkpoint requires multiple cyclin types. One way to explain this is based on the distinct subcellular localizations of mitotic cyclins. Once activated, the checkpoint can operate in multiple cellular compartments, such as the nucleus and the cytoplasm. Although signals coordinate entry into mitosis in the cytoplasm and nucleus, persistent nuclear checkpoint activity can prevent mitotic entry despite cytoplasmic Cdk activity. Individual cyclins would not be able to act on their own to reverse the checkpoint in all compartments if each is excluded from one compartment. For example, cyclin B is efficiently excluded from the nucleus in cycle 13 embryos and presumably would not contribute to checkpoint reversal in this compartment, whereas cyclin B3 is nuclear. In embryos with only CycB, the checkpoint should be reversed first in the cytoplasm; however, progress to mitosis should depend on slower reversal in the nucleus, which might be based on communication between compartments. Consistent with this proposal, injection of CycB protein has been shown to preferentially drive cytoplasmic, but not nuclear, mitotic events. The full complement of cyclins with distinct localizations, however, appears to reverse the checkpoint promptly and coordinately in all the compartments (Yuan, 2012).

These data demonstrate a cyclin-type effect on reversal of the DNA replication checkpoint, which emphasizes the qualitatively distinct contributions among mitotic cyclins during mitotic entry. This study opens many further questions, such as what causes the checkpoint to inactivate? How do mitotic cyclins promote checkpoint reversal? It is believed that answers to these questions will lead to a fuller understanding of the timing mechanism of the cell division cycle (Yuan, 2012).

Contributions of DNA repair, cell cycle checkpoints and cell death to suppressing the DNA damage-induced tumorigenic behavior of Drosophila epithelial cells

When exposed to DNA-damaging agents, components of the DNA damage response (DDR) pathway trigger apoptosis, cell cycle arrest and DNA repair. Although failures in this pathway are associated with cancer development, the tumor suppressor roles of cell cycle arrest and apoptosis have recently been questioned in mouse models. Using Drosophila epithelial cells that are unable to activate the apoptotic program, evidence is provided that ionizing radiation (IR)-induced DNA damage elicits a tumorigenic behavior in terms of E-cadherin delocalization, cell delamination, basement membrane degradation and neoplasic overgrowth. The tumorigenic response of the tissue to IR is enhanced by depletion of Okra/DmRAD54 or spnA/DmRAD51-genes required for homologous recombination (HR) repair of DNA double-strand breaks in G2-and it is independent of the activity of Lig4, a ligase required for nonhomologous end-joining repair in G1. Remarkably, depletion of Grapes/DmChk1 or Mei-41/dATR-genes affecting DNA damage-induces cell cycle arrest in G2-compromises DNA repair and enhances the tumorigenic response of the tissue to IR. On the contrary, DDR-independent lengthening of G2 has a positive impact on the dynamics of DNA repair and suppressed the tumorigenic response of the tissue to IR. These results support a tumor suppressor roles of apoptosis, DNA repair by HR and cell cycle arrest in G2 in simple epithelia subject to IR-induced DNA damage (Dekanty, 2014).


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grape: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 15 April 2014

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