The ability to respond to DNA damage and incomplete replication ensures proper duplication and stability of the genome. Two checkpoint kinases, ATM and ATR, are required for DNA damage and replication checkpoint responses. In Drosophila, the ATR ortholog (MEI-41) is essential for preventing entry into mitosis in the presence of DNA damage. In the absence of MEI-41, heterozygosity for the E(mus304) mutation causes rough eyes. We found that E(mus304) is a mutation in DNApol-180, which encodes the catalytic subunit of DNA polymerase . We did not find any defects resulting from reducing Pol by itself. However, reducing Pol in the absence of MEI-41 resulted in elevated P53-dependent apoptosis, rough eyes, and increased genomic instability. Reducing Pol in mutants that lack downstream components of the DNA damage checkpoint (DmChk1 and DmChk2) results in the same defects. Furthermore, reducing levels of mitotic cyclins rescues both phenotypes. We suggest that reducing Pol slows replication, imposing an essential requirement for the MEI-41-dependent checkpoint for maintenance of genome stability, cell survival, and proper development. This work demonstrates a critical contribution of the checkpoint function of MEI-41 in responding to endogenous damage.

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EUKARYOTIC cells constantly experience exogenous DNA damage from the environment as well as endogenous damage that occurs during DNA metabolism and replication. An inability to respond to either type of damage can result in genomic instability and loss of genetic material. To maintain genomic stability, cells have developed mechanisms for responding to DNA damage and/or incomplete replication. Maintenance of genome stability can be accomplished by coupling replication and repair with cell cycle regulation via the DNA damage checkpoint pathway. In this pathway, sensors recognize incomplete replication and/or DNA damage and then stimulate a variety of responses, including phosphorylation of downstream transducers. These transducers then activate or inactivate effectors that directly affect cell cycle progression, resulting in cell cycle arrest, presumably to allow time to complete replication or repair the damage (reviewed in SANCAR et al. 2004).

ATM (for ataxia telangiectasia mutated) and ATR (for ATM and Rad3 related) are two kinases that mediate the DNA damage checkpoint in response to incomplete replication and DNA damage. These kinases are highly conserved and required for G₁-S, intra-S, and G₂-M checkpoint responses (reviewed in SANCAR et al. 2004; reviewed in SHILOH 2003). ATM and ATR function upstream of conserved transducers of the checkpoint response, Chk1 and Chk2. In mammals, ATM primarily phosphorylates Chk2 in response to damage that results in double-strand breaks (DSBs) (CANMAN et al. 1998). In contrast, ATR primarily activates Chk1 in response to incomplete replication and/or damage that results in single-strand DNA (CLIBY et al. 1998; WRIGHT et al. 1998; UNSAL-KACMAZ et al. 2002; DAS and DASHNAMOORTHY 2004). Although there is some functional overlap of these kinases and the transducers of the checkpoint response, the ATR/Chk1 pathway is primarily responsible for the intra-S checkpoint (BODDY et al. 1998; CHEN and SANCHEZ 2004; HELT et al. 2005; reviewed in SANCHEZ et al. 1996; SANCAR et al. 2004).

Many studies have characterized DNA damage response pathways using exogenous sources of damage, such as hydroxeurea, UV, ionizing radiation (IR), and alkylating agents. However, it is presumed that the most common type of damage that a cell must respond to is endogenous, such as lesions that occur during replication and regular DNA metabolism (LINDAHL 1993; BISHOP et al. 2000; FROSINA 2000). Evidence from other organisms indicates that orthologs of ATR have important roles in responding to endogenous damage. Cells from ATR-Seckel syndrome patients with a mutated form of ATR demonstrate elevated genome damage and chromosome breaks following replication stress (O'DRISCOLL et al. 2004) and ATR-deficient mouse cells also accumulate spontaneous chromosomal breaks (BROWN and BALTIMORE 2003). Similarly, Saccharomyces cerevisiae mutants lacking the ATR ortholog Mec1 have elevated rates of gross chromosomal rearrangements (COBB et al. 2005) as well as spontaneous DNA breaks that map to replication slow zones (CHA and KLECKNER 2002). These results demonstrate the need to further understand how ATR responds to endogenous damage that occurs during DNA synthesis.

The role of ATR in response to endogenous damage has been investigated in multiple organisms by examining interactions between checkpoint proteins and components of the replication machinery, especially DNA polymerase (Pol) (reviewed in FOIANI et al. 1997). Initiation of replicative DNA synthesis begins with formation of an RNA primer by primase. Pol forms a complex with primase and is responsible for synthesizing the initial DNA extension from the primer. Thus, Pol is required to initiate both leading-strand and lagging-strand synthesis; however, Pol is required continuously for lagging-strand synthesis, since every Okazaki fragment initiates with an RNA primer. In S. cerevisiae, Pol is stabilized in a Mec1-dependent manner after treatment with the replication inhibitor hydroxyurea (HU) (COBB et al. 2003), and decreasing expression of the catalytic subunit of Pol by 90% in a mec1 mutant results in increased genomic instability (LEMOINE et al. 2005). In Schizosaccaromyces pombe, temperature-sensitive mutants of pol cause activation of Chk1 (D'URSO et al. 1995; BHAUMIK and WANG 1998). In Xenopus laevis, uncoupling of helicase and polymerase activity during replication results in Pol-dependent activation of Chk1 (BYUN et al. 2005; CORTEZ 2005). These results reveal a conserved genetic interaction between DNA Pol and the ATR-mediated damage response.

Drosophila ATR, encoded by mei-41, is the primary kinase required for the checkpoint response after DNA damage during all phases of the cell cycle (HARI et al. 1995; SIBON et al. 1999; BRODSKY et al. 2000; GARNER et al. 2001; JAKLEVIC and SU 2004; BI et al. 2005; LAROCQUE et al. 2007). mei-41 mutants are sensitive to a wide range of agents that damage DNA or inhibit DNA replication, including ultraviolet light, methyl methanesulfonate, IR, and HU (BOYD et al. 1976; SIBON et al. 1999). Sensitivity to this broad spectrum of damaging agents suggests that MEI-41-mediated checkpoints are essential in the response to many types of DNA damage throughout the cell cycle. As in mice, humans, and S. cerevisiae, mei-41 mutants have an elevated frequency of spontaneous chromosome breaks (GATTI 1979; BAKER et al. 1980; BANGA et al. 1986).

To learn more about the role of the ATR-mediated cell cycle checkpoint in responding to replication defects, we genetically reduced Pol in mei-41 mutants. This resulted in P53-dependent apoptosis, increased genomic instability, and P53-dependent morphological defects. Our data also suggest that cell cycle regulation by MEI-41 is the major component of this interaction, although loss of the Chk1- and Chk2-dependent checkpoint cannot completely account for the defects.

Drosophila stocks and genetics:

Flies were maintained on standard medium at 25°. The mei-41 mutant males were hemizygotes of mei-41^29D (LAURENCON et al. 2003). The cyclin mutations used were CycA^C8LR1 (SIGRIST and LEHNER 1997) and CycB² (JACOBS et al. 1998). The lok mutants were homozygous for lok³⁰ and the grp mutants were heteroallelic for grp²⁰⁹ and grp^Z5170 (LAROCQUE et al. 2007). The p53 mutants used were p53^5A-1-4 (RONG et al. 2002). Reductions in Pol used the E(mus304) mutant chromosome (BRODSKY et al. 2000). Recombinants of E(mus304) and p53^5A-1-4 were generated and verified using allele-specific PCR for both mutations and for presence of a rough-eye phenotype in mei-41 mutants.

Mapping mutations in DNApol-180:

Recombination mapping between ebony (e) and claret was used to confirm the published location of E(mus304) (BRODSKY et al. 2000) using the rough eyes in mei-41 mutants as the phenotypic marker. Deficiencies of the area surrounding and including 89D-F were used to narrow the location of the region down to five genes: E2f, CG31176, CG6353, CG15497, and DNApol-180. Two genes, E2f and CG31176, were excluded from consideration when mei-41 mutants failed to have a rough-eye phenotype when heterozygous for these mutations. The E(mus304) chromosome was sequenced for changes in pol. Using GFP selection, genomic DNA was prepared from single embryos homozygous for E(mus304) and PCR was performed using gene-specific primers. PCR reactions contained 10 mM Tris–HCl, pH 9.0, 50 mM KCl, 2.5 mM MgCl₂, 0.1% Triton X-100, 1.25 µM of each primer, 250 µM each dNTP, 2 µl of the genomic DNA prep, and Taq DNA polymerase in a 20-µl volume. PCR products were isolated using gel electrophoresis, purified, and sequenced directly. The mutation was confirmed by sequencing the opposite strand. Mutations found from the ethyl methanesulfonate (EMS) screen were confirmed this way as well.

EMS mutagenesis:

One- to 3-day-old males were fed 25 mM EMS (Fluka Chemika) in 1% sucrose on cotton pads overnight. Males were then transferred to clean bottles for 1 day and then crossed to mei-41/FM7 females in bottles. To avoid screening progeny resulting from mutagenesis of premeiotic germline cells, males were discarded after 5 days. F₁ male progeny mutant for mei-41 were screened for rough eyes, indicative of a possible dominant autosomal mutation that interacts with the mei-41 mutation. Mutations that mapped to chromosome 3 were crossed to E(mus304), and those that failed to complement the homozygous lethality phenotype of E(mus304) were sequenced to find mutations in DNApol-180.

SEM imaging:

Adult fly heads were fixed in phosphate-buffered saline (PBS) and 4% paraformaldehyde. Samples were stored at 4° for several days before being dehydrated through a series of washes in increasing ethanol concentration, with a final rinse in 100% ethanol, and then prepped with assistance from the Microscopy Services Laboratory at the University of North Carolina-Chapel Hill. Samples were transferred in absolute ethanol to a Balzers CGD 020 critical point dryer (BAL-TEC, Balzers, Principality of Liechtenstein) and dried using liquid CO₂ as the solvent solution. Heads were mounted and sputter coated with gold:palladium alloy (60:40) using a Hummer X Sputter Coater (Anatech, Alexandria, VA). Specimens were viewed on a Cambridge Stereoscan S200 scanning electron microscope (LEO Electron Microscopy, Thornwook, NY) using an acceleration voltage of 20 kV and a working distance of 25 mm.

Detecting apoptotic cells:

Imaginal discs were dissected from third instar larvae of appropriate genotypes in Ringer's solution and fixed for 45 min in 4% formaldehyde and PBS with 0.1% Triton-X (PBT). Discs were washed and blocked in PBT with 5% bovine serum albumin. Discs were incubated with 1:500 dilution of rabbit anti-human cleaved caspase-3 (Asp175) antibody (Cell Signaling Technology) in PBT overnight at 4°. Discs were incubated for 2 hr at room temperature with 1:1000 secondary goat anti-rabbit rhodamine-conjugated antibody (Molecular Probes, Eugene, OR) or secondary goat anti-rabbit fluorescein-conjugated antibody (Molecular Probes), stained with 10 µg/ml DAPI in PBT, and mounted with Flouromount-G (Southern Biotechnology Associates). Discs were visualized using TRIT-C and FIT-C filters of a Nikon Eclipse E800 fluorescent microscope. Quantification was performed on images of 7–14 wing discs of each genotype. Each disc was counted for the total number of caspase-positive cells per disc to obtain an average. Significance was computed using an unpaired t-test with Welch's correction using InStat statistical software.

Genomic instability phenotypes:

Loss of heterozygosity (LOH) at multiple wing hair (mwh) was detected as described by BRODSKY et al. (2000). Briefly, wings of appropriate genotype were dehydrated in isopropanol and mounted in 1:1 methylsalicilate:Canada balsam (Sigma, St. Louis). Each wing was viewed at x40 using the light filter of a Nikon Eclipse E800 fluorescent microscope and scored for mwh phenotype. A total of 10–20 wings were examined for each genotype to obtain an average rate of mitotic clones per wing. Standard deviations were determined on the basis of averages; significance was computed using an unpaired t-test with Welch's correction using InStat statistical software.

To detect increases in mitotic crossovers, unbalanced single males of appropriate genotypes heterozygous for ebony and scarlet (st) were crossed to ru h th st cu sr e Pr ca/TM6B females. Crossovers between st and e in the premeiotic male germline were scored in progeny of this cross. Over 3000 progeny were scored for each genotype. Significance was determined by analyzing a contingency table using chi-square approximation with Yates correction available through InStat statistical program.

Enhancer of mus304 is an allele of DNApol-180:

A previously published study reported a spontaneous mutation that interacts genetically with mei-41 and mus304, which encodes the ortholog of ATR-IP (BRODSKY et al. 2000). This mutation, referred to as Enhancer of mus304, is homozygous embryonic lethal (data not shown). However, heterozygosity for this mutation in mei-41 or mus304 mutants results in a rough-eye phenotype. The Enhancer of mus304 mutation was mapped to region 93F on the third chromosome (BRODSKY et al. 2000). We further mapped Enhancer of mus304 (see MATERIALS AND METHODS) to a region that includes DNApol-180, which encodes the catalytic subunit of Pol. We sequenced the DNApol- 180 coding region from the mutant chromosome and found a deletion of a single base pair in the third exon at codon 301 (Figure 1). This deletion results in a frameshift and a premature stop 29 codons downstream.

View larger version (6K): In this window In a new window Download PPT slide   FIGURE 1.— Enhancer of mus304 is an allele of DNApol-180. E(mus304) was roughly mapped and predicted to be a mutation in DNApol-180 (see MATERIALS AND METHODS). Sequencing of this region confirmed a loss of an "A" in the third exon at codon 301, resulting in a frameshift and a premature stop 29 codons downstream. An EMS mutagenesis and screen for mutations conferring rough eyes to mei-41 mutants resulted in two new alleles (see MATERIALS AND METHODS); both were nonsense mutations in glutamine codons. Shaded boxes are coding exons. Mutations are marked with asterisks.

 
To confirm that the interaction with mei-41 is due to a mutation in DNApol-180, we conducted a mutagenesis screen to identify mutations that caused rough eyes when heterozygous in a mei-41 mutant (see MATERIALS AND METHODS). Two new alleles of DNApol-180 were recovered (Figure 1). Both are nonsense mutations at glutamine codons (1132 and 1322). Heterozygosity for any of these alleles, or for a deletion of this region, confers a rough-eye phenotype to mei-41 mutants. We conclude that reducing the dosage of DNApol-180 (hereafter referred to as pol) by half is sufficient to cause a developmental defect in mei-41 mutants.

Reducing Pol in mei-41 mutants causes an increase in cell death:

The Drosophila compound eye comprises 800 ommatidia, each of which has a precise number of cells in an identical arrangement, resulting in a smooth appearance. The correct number of cells results from a carefully orchestrated sequence in which some cells differentiate and others undergo cell death (reviewed in BONINI and FORTINI 1999). Because of this, eye development is highly sensitive to changes in cell survival, unlike other adult organs, such as the wing, whose cell number is largely dispensable for development (BAKER 2001). For example, overexpression of P53, which is required for DNA-damage-induced apoptosis, disrupts formation of an ordered array of ommatidia, resulting in eyes with a rough appearance (OLLMANN et al. 2000; LEE et al. 2003). Other mutations have also revealed a correlation between increased apoptosis and rough eyes. Temperature-sensitive mutations in the tefu gene, which encodes Drosophila ATM, cause both rough eyes and increased apoptosis in imaginal discs (SILVA et al. 2004).

To determine whether mei-41 mutants that are heterozygous for a pol mutation have increased apoptosis in proliferating imaginal disc cells, we quantified the number of apoptotic cells per imaginal wing disc, using an antibody raised against human-activated caspase-3, a conserved effector caspase that is cleaved and subsequently activated during apoptosis (reviewed in VAN LANCKER 2006). The human cleaved caspase-3 antibody also recognizes Drosophila cells undergoing DNA-damage-induced apoptosis (GIRALDEZ and COHEN 2003). The average number of apoptotic cells was increased fourfold in mei-41 mutants compared to wild-type larvae (P < 10^–5; Figure 2C). A similar increase was also seen in mus304 mutants (P < 10^–5; data not shown). Heterozygosity for a pol mutation did not increase apoptosis by itself (P = 0.08), but led to a further increase in mei-41 mutants (P < 10^–5 for mei-41; pol/+ compared to mei-41 alone; Figure 2, A and C). Similar results were seen in other imaginal discs and when staining with the vital dye acridine orange (data not shown). These observations show that reducing Pol in mei-41 or mus304 mutants causes increased apoptosis in proliferating tissues. Most imaginal tissues can compensate for increased cell death through increased proliferation (HAYNIE and BRYANT 1977; JAKLEVIC and SU 2004), so development of most adult appendages appears to be unaffected. Patterning of the compound eye, however, is exquisitely sensitive to changes in cell survival; as a result, the rough-eye phenotype is a sensitive indicator of increased cell death.

View larger version (77K): In this window In a new window Download PPT slide   FIGURE 2.— Reducing Pol in mei-41 mutants results in a variety of phenotypes. (A) Wing discs of third instar larvae were dissected, fixed, and stained with an antibody to cleaved human caspase 3, marking apoptotic cells. (B) As shown previously (BRODSKY et al. 2000), mei-41; pol/+ mutants have a rough-eye phenotype that includes fused ommatidia and tissue loss. mei-41 mutants are indistinguishable from wild type and are used for comparison. This rough-eye phenotype of mei-41; pol/+ mutants was rescued by eliminating P53. (C) Quantification of apoptosis phenotype demonstrated in A. mei-41 mutants had an increase in apoptosis compared to wild type (P < 10–6), and this was quantitatively more severe when Pol was reduced (P < 10–4 when compared to mei-41). Mutations in p53 restored apoptosis to the levels seen in mei-41 single mutants (P = 0.19 compared to mei-41).

 

The increased apoptosis and rough-eye phenotypes of mei-41; pol/+ mutants are P53 dependent:

As noted earlier, previous studies have suggested a correlation between P53-dependent apoptosis and eye development. Overexpression of P53 causes a rough-eye phenotype (OLLMANN et al. 2000; LEE et al. 2003), similar to the phenotype that we observe when Pol is reduced in mei-41 mutants (BRODSKY et al. 2000; Figure 2A). We hypothesized that reducing Pol in mei-41 mutants elicits a P53-dependent apoptotic response, leading to a rough-eye phenotype. To test this hypothesis, we eliminated P53 expression in these mutants. Loss of P53 in mei-41; pol/+ mutants completely rescued the rough-eye phenotype (Figure 2B) and restored the level of apoptosis to that seen in mei-41 single mutants (Figure 2, A and C). Together, these data indicate that reducing Pol results in damage that elicits a MEI-41-dependent DNA damage response. In the absence of MEI-41, proliferating cells with reduced Pol undergo P53-dependent apoptosis, resulting in cell death and misregulated development of the adult eye.

mei-41; pol/+ mutants have increased genomic instability:

An inability to respond to spontaneous damage leads to increased genomic instability in mei-41 and mus304 mutants (BAKER et al. 1978; GATTI 1979; BRODSKY et al. 2000). One manifestation of genomic instability is increased LOH; both mei-41 and mus304 mutants have increased LOH at the mwh locus (BAKER et al. 1978; BRODSKY et al. 2000). We tested whether decreasing Pol in mei-41 mutants results in a further increase in LOH frequency. We found an increase in LOH in mei-41 mutants relative to wild type (P < 10^–5; Figure 3), as shown previously. There was no increase in pol/+ mutants relative to wild type (P = 0.83), but heterozygosity for pol resulted in an increase in LOH in mei-41 mutants (P < 10^–5, relative to mei-41 single mutants).

View larger version (48K): In this window In a new window Download PPT slide   FIGURE 3.— Reducing Pol in mei-41 mutants results in an increase of LOH. mwh mutant flies have multiple hairs from each hair cell of the adult wing, and mwh/+ flies are phenotypically normal. LOH at mwh will result in clones of cells with multiple hairs per cell (circled). LOH can occur through spontaneous mutation, gene conversion, deletion, or mitotic crossing over. Individual adult wings were scored for mwh clones. Bars represent the average number of clones per wing, and lines are the standard deviation based on 10–12 wings/genotype. Significance was determined by an unpaired t-test with Welch's correction.

 
LOH can result from many mechanisms, including chromosome loss, deletion, spontaneous mutation, and mitotic crossing over (reviewed in PÂQUES and HABER 1999). We quantified the frequency of mitotic crossovers between two markers on the third chromosome, e and st. Mitotic crossovers that occur in premeiotic germline cells are scored in progeny of males. As seen in wild-type, mei-41 and pol/+ mutants completely lacked mitotic crossovers between these markers. In contrast, when Pol was reduced in mei-41 mutants, there was a significant increase in the frequency of mitotic crossovers (P < 0.05; Table 1). This suggests that a subset, if not all, of the increased LOH observed at the mwh locus can be attributed to an increase in mitotic crossovers.

View this table: In this window In a new window   TABLE 1 Mitotic crossovers between ebony and scarlet

 

Phenotypes manifested in mei-41; pol/+ mutants can be rescued by reducing mitotic cyclins:

We hypothesize that reducing Pol levels elicits a DNA damage response, due either to slowed and/or incomplete replication or to uncoupling of leading- and lagging-strand synthesis. We propose that this collective replication stress requires a MEI-41-dependent checkpoint response to regulate cell cycle progression, perhaps by giving enough time to complete replication before entry into mitosis. To test this hypothesis, we sought to bypass the requirement for MEI-41 by delaying entry into mitosis through other means. Reducing the maternal contribution of the mitotic cyclins, cyclin A and cyclin B, slows early embryonic cell cycle progression (EDGAR et al. 1994). Reducing cyclin A and cyclin B also bypasses the requirement for MEI-41 in regulating the midblastula transition during early embryonic development (SIBON et al. 1999) and rescues the sensitivity of mei-41 mutants to P-element excision (LAROCQUE et al. 2007).

We attempted to rescue the rough-eye phenotype in mei-41; pol/+ mutants by reducing cyclin A and/or cyclin B. Cyclin B reduction partially rescued this phenotype, and reducing cyclin A (or both cyclin A and cyclin B) completely rescued the rough-eye phenotype, resulting in eyes that were indistinguishable from those of mei-41 mutants or wild-type flies (Figure 4A). We then asked if we could rescue the increased-apoptosis phenotype by reducing mitotic cyclins. Similar to the rescue of the rough-eye phenotype, reducing cyclin A in these mutants rescued levels of apoptosis that were indistinguishable from mei-41 single mutants (P = 0.14) or mei-41; CycA/+ mutants (P = 0.54; Figure 4C). These data demonstrate that mitotic cyclin reduction is capable of suppressing both apoptosis and rough eyes, supporting the idea that reducing Pol elicits a damage response that requires the checkpoint function of MEI-41 to regulate cell cycle progression.

View larger version (84K): In this window In a new window Download PPT slide   FIGURE 4.— Reducing mitotic cyclins rescues the rough-eye phenotype and apoptosis of mei-41; pol/+ mutants. (A) The rough-eye phenotype of mei-41; pol/+ mutants is partially rescued by reducing CycB and completely rescued when CycA is reduced. (B and C) The apoptosis phenotype observed in mei-41; pol/+ mutants is rescued by reducing CycA. Samples were prepared and scored as described in Figure 2. Bars represent averages of 7–10 imaginal wing discs/genotype, and lines represent standard deviations. Significance was determined by an unpaired t-test with Welch's correction.

 

Loss of the GRP/LOK-mediated checkpoint accounts for a degree of the phenotypes observed in mei-41; pol/+ mutants:

Rescue of rough eyes and apoptosis by mitotic cyclin reduction suggests that cell cycle regulation contributes to the phenotypes that we have reported here. To further test this hypothesis, we examined the effects of Pol reduction on loss of Chk1 and Chk2, which have partially redundant roles in mediating the DNA damage checkpoint response in mammals (BODDY et al. 1998; CHEN and SANCHEZ 2004; HELT et al. 2005; reviewed in SANCHEZ et al. 1996; SANCAR et al. 2004). The Drosophila orthologs of Chk1 and Chk2 are encoded by grp and lok, respectively. Like mei-41 mutants, grp lok mutants are completely defective in the replication and damage checkpoints (SU et al. 1999; YU et al. 2000; MASROUHA et al. 2003; BRODSKY et al. 2004; JAKLEVIC and SU 2004; DE VRIES et al. 2005; ROYOU et al. 2005; LAROCQUE et al. 2007).

We first examined grp and lok mutants for the rough-eye phenotype conferred by heterozygosity for a pol mutation. The eyes of lok; pol/+ flies were indistinguishable from those of wild-type flies. In contrast, grp; pol/+ mutant males had a rough-eye phenotype (Figure 5A), but females had wild-type eyes. In grp lok; pol/+ mutants, both males and females exhibited rough eyes; however, the phenotype was still not as severe as that of mei-41; pol/+ mutants. We also measured the effects of grp and lok mutations on apoptosis. Neither the single mutants (grp or lok) nor the grp lok double mutants had the elevated levels of apoptosis observed in mei-41 mutants (Figure 5B). However, lok; pol/+ mutants had a slight increase in apoptosis compared to wild type (P < 0.05), and grp; pol/+ and grp lok; pol/+ mutants had a more substantial increase compared to wild type (P < 10^–4). Interestingly, the levels of apoptosis in these mutants were not as high as in mei-41; pol/+ mutants (P < 10^–4). These data demonstrate that the Chk1/Chk2-meditated checkpoint function of MEI-41 plays an important role in response to reducing Pol; however, the intermediate phenotypes suggest that loss of this checkpoint cannot completely account for the severity of mei-41; pol/+ mutants.

View larger version (93K): In this window In a new window Download PPT slide   FIGURE 5.— Analysis of grp and lok mutants when Pol is reduced. (A) While eyes of lok; pol/+ mutants are indistinguishable from those of wild-type flies, grp single mutants and grp lok double mutants have rough eyes when Pol is reduced. (B and C) lok; pol/+ have only a slight increase in apoptosis compared to wild type (P < 0.05) whereas grp; pol/+ and grp lok; pol/+ mutants have a greater increase in apoptosis (P < 10–4) but are not significantly different from each other (P = 0.16). Samples were prepared as described in Figure 2. Bars represent averages of 7–14 imaginal wing discs/genotype, and lines represent standard deviations. Significance was determined by an unpaired t-test with Welch's correction.

 

We have shown here that genetically reducing Pol levels by only half in mei-41 mutants results in increased P53-dependent apoptosis, rough eyes, and genomic instability, including elevated mitotic crossing over. Reducing mitotic cyclin levels rescues at least some of these phenotypes, supporting the idea that loss of MEI-41-dependent cell cycle regulation contributes greatly to the defects. However, the GRP/LOK-mediated checkpoint does not account for the severity of phenotypes observed in mei-41 mutants. We suggest here that reducing Pol results in P53-inducing damage, such as incomplete replication, stalled replication forks, or uncoupling of leading- and lagging-strand synthesis. This "replication stress" requires the checkpoint function of MEI-41 to maintain developmental processes, cell survival, and genomic stability.

Reducing Pol alone does not cause any detectable defects, which suggests that the damage caused by reducing Pol in an otherwise wild-type background is relatively mild. It is possible that the defects that we observed are an additive effect of defects in mei-41 mutants that we did not detect in pol/+ flies. A more likely explanation is that MEI-41 function is exceptionally important in responding to the very low level of endogenous damage that results from reducing Pol. This interpretation is consistent with our previous finding that mei-41 mutants have reduced viability when a single P element is undergoing transposition during development (LAROCQUE et al. 2007).

If the damage that results from reducing Pol requires the checkpoint response of MEI-41, it should elicit a checkpoint response in animals that are wild type for mei-41. It is difficult to detect S-phase checkpoints in Drosophila tissues, but there has been one report of MEI-41-dependent decrease in BrdU incorporation into larval neuroblasts, following treatment with 1600 rad of IR (JAKLEVIC and SU 2004). We were unable to detect this decreased BrdU incorporation in pol/+ larvae or in wild-type larvae after irradiation (data not shown). In contrast, irradiation induces a robust MEI-41-dependent delay of entry into mitosis (HARI et al. 1995). In imaginal discs, this G₂-M checkpoint is readily detected by staining with a marker for mitotic cells after irradiation with as little as 500 rad (BRODSKY et al. 2000; BI et al. 2005; LAROCQUE et al. 2007); we were unable to detect any effect of reducing Pol on the number of mitotic cells (data not shown). A likely explanation is that irradiation induces a burst of damage, resulting in rapid cessation of entry in mitosis that can be detected soon after treatment, whereas any damage resulting from genetic reduction of Pol would occur and be repaired throughout development. It might be possible to detect an increased steady-state level of MEI-41-dependent phosphorylation of checkpoint transducers or effectors, but this would depend on the level of damage resulting from reduction of Pol.

Reducing other components of the Pol complex did not result in rough eyes in mei-41 mutants. These included the primase subunit (DNAprim), and the 50- and 73-kDa subunits of Pol (data not shown). We also reduced levels of other replicative polymerases and components of replication, using null alleles and/or deficiencies of DNApol-, DNApol, E2f, and mus209, which encodes PCNA. None of these manipulations caused rough eyes in mei-41 mutants. It is possible that there is an unknown function of Pol responsible for the interactions with MEI-41. Alternatively, whereas pola-180 mutants are embryonic lethal, the reported lethal phenotypes of DNAprim, E2f, and PCNA null mutants include survival to at least first instar larvae (ROYZMAN et al. 1997; CHEN et al. 2000; HENDERSON et al. 2000). The 180-kDa catalytic subunit therefore may be the limiting factor of the primase complex, and reduction of this subunit, as observed with embryonic lethality, may have a more profound effect on replication than reducing other components of the replication machinery.

Previous work has demonstrated a link between increased apoptosis and rough-eye phenotypes (OLLMANN et al. 2000; LEE et al. 2003; SILVA et al. 2004). We therefore tested imaginal discs to see whether or not there was an increase in apoptosis that could presumably lead to rough eyes. We found a strong correlation between rough eyes and an increase in apoptosis: genotypes that had rough eyes also had an increase in apoptosis, and reducing the number of apoptotic cells also rescues rough-eye phenotypes (mei-41; CycA/pol). We directly tested whether eye development was dependent on P53-mediated apoptosis and found that eliminating P53 completely rescues both apoptosis and the rough-eye phenotype. Some genotypes that had an increase in apoptosis compared to wild type did not result in rough eyes: mei-41 single mutants, mus304 single mutants, and grp; pol/+ mutant females (data not shown). Overall, however, we found a strong correlation between two dramatic phenotypes associated with mutants in cell cycle regulation and in reducing Pol.

Reducing mitotic cyclin levels rescued the rough-eye phenotype and increased apoptosis of mei-41; pol/+ mutants. We propose that reducing cyclins slows cell cycle progression and therefore eliminates the need for MEI-41 checkpoint function to respond to damage induced by reducing Pol. We do not know the effects of cyclin reduction on cell cycle timing in proliferating cells of imaginal discs, but reducing cyclins does affect cell cycle timing during embryogenesis (EDGAR et al. 1994; CREST et al. 2007) and ameliorates DSB repair defects of mei-41 mutants (LAROCQUE et al. 2007). It is possible that mitotic cyclin reduction has no effect on response to DNA damage, but contributes to proper development by regulating developmentally controlled apoptosis. We cannot directly test this possibility, but our finding that the apoptosis and rough-eye phenotypes are P53 dependent supports the proposal that reducing Pol elicits a DNA damage response, since P53 is required for damage-induced apoptosis but not for developmentally regulated programmed cell death (LEE et al. 2003; BRODSKY et al. 2004; JAKLEVIC and SU 2004; this study).

The interactions among grp, lok, and pol suggest varying contributions of GRP and LOK to the phenotypes reported here. The lok; pol/+ mutants were indistinguishable from wild type in eye development and had only slight increases in apoptosis. In contrast, in grp; pol/+ and grp lok; pol/+, there was a dramatic increase in apoptosis. These genotypes differed from one another in that only males had rough eyes in the grp; pol/+ mutants, but both sexes had rough eyes in the grp lok; pol/+ mutants. It is not clear why there is a difference between males and females in grp; pol/+ mutants, as different genetic backgrounds show a similar discrepancy. It is possible that the severity of the defect in grp; pol/+ mutants is near the threshold for causing rough eyes and that this threshold is lower in males than in females. Comparing all three genotypes (grp and lok single mutants and grp lok double mutants), however, we conclude that the phenotypic effects of reducing Pol can be attributed predominantly to the GRP-mediated checkpoint. Nonetheless, there does appear to be some redundancy between GRP and LOK in these assays. Partial redundancy between Chk1 and Chk2 has been demonstrated in other organisms (BODDY et al. 1998; CHEN and SANCHEZ 2004; HELT et al. 2005; reviewed in SANCHEZ et al. 1996; SANCAR et al. 2004), as well as in the DNA damage checkpoint response in Drosophila (XU et al. 2001; BRODSKY et al. 2004; LAROCQUE et al. 2007) and in repair of DSBs induced through P-element excision (LAROCQUE et al. 2007).

Loss of both GRP and LOK did not produce defects as severe as those observed when MEI-41 was absent. We conclude that the GRP/LOK-mediated checkpoint cannot completely account for the defects seen in mei-41; pol/+ flies. Studies in mammalian cells suggest a checkpoint-independent role for mammalian ATM kinases in DNA repair (reviewed in JEGGO et al. 1998; LOBRICH and JEGGO 2005; JEGGO and LOBRICH 2006; O'DRISCOLL and JEGGO 2006). We previously showed that mei-41 mutants are more sensitive to P-element excision and have more severe defects in homologous recombinational repair compared to grp lok double mutants (LAROCQUE et al. 2007), and JAKLEVIC and SU (2004) found that mei-41 mutants are killed by doses of IR that are not lethal to grp mutants, even though both mutants are defective in the IR-induced G₂-M checkpoint. OIKEMUS et al. (2006) found that both spontaneous and IR-induced chromosome breaks were increased in mei-41 mutants but not in grp lok double mutants, suggesting that MEI-41 has a role in preventing chromosome breaks that is independent of GRP and LOK. Together, these studies strongly suggest that there is a role of MEI-41 that is independent of the GRP/LOK-mediated checkpoint in response to reducing Pol.

Despite numerous observations that MEI-41 has GRP/LOK-independent functions in response to DNA damage, it is possible that the MEI-41-mediated checkpoint is not completely eliminated in grp lok mutants and that there is an unidentified transducer of the checkpoint pathway. We and others have not been able to detect a G₂-M checkpoint after IR in grp lok mutants (LIU et al. 2000; XU et al. 2001; BRODSKY et al. 2004; LAROCQUE et al. 2007), consistent with studies in other model organisms that indicate that ATR/ATM-dependent DNA damage checkpoints are transduced entirely through Chk1 and Chk2 (BODDY et al. 1998; CHEN and SANCHEZ 2004; reviewed in SANCHEZ et al. 1996; SANCAR et al. 2004). We therefore favor the alternative hypothesis that MEI-41 has some role independent of its checkpoint function in response to damage caused by reducing Pol.

In conclusion, we have identified an interaction between regulators of the cell cycle and a component of replication machinery. These interactions are necessary for proper development of adult organs, maintaining genomic stability, and regulation of cell survival. This study reveals a checkpoint-dependent response when Pol is reduced, suggesting the importance for development and cell survival in responding to endogenous damage that occurs during normal DNA metabolism. Previous work in fungi and humans highlights a role for ATR orthologs in maintaining fragile site stability in response to slowing replication by aphidicolin treatment or genetically reducing Pol (CASPER et al. 2002; LEMOINE et al. 2005). Additionally, work in Xenopus has demonstrated that uncoupling of DNA polymerases from MCM helicase via aphidicolin treatment (WALTER and NEWPORT 2000), cis-platinum treatment, or UV irradiation (BYUN et al. 2005) activates the ATR-dependent checkpoint. While most checkpoint studies rely on exogenously induced damage, our findings reveal the importance of an ATR-mediated checkpoint in responding to relatively mild endogenous defects. The results reported here demonstrate yet another conserved interaction between cell cycle checkpoint response and replication machinery, two cellular processes that are integral for genomic stability and cell survival.

We thank Shawn Ahmed and members of the Sekelsky lab for thoughtful discussions, Manfred Frasch for stocks, and the Duronio lab for antibodies and reagents. We also thank Victoria Madden at the Microscopy Services Laboratory at the University of North Carolina (UNC)-Chapel Hill for assistance with SEM sample preparation and imaging. J.R.L. was supported by a Graduate Research Fellowship from the National Science Foundation. S.K.H. was supported by the UNC-Chapel Hill Office of Undergraduate Research and the Frances C. and William P. Smallwood Foundation. This research was supported by a grant from the American Cancer Society (RSG-05-138-01-GMC) to J.S.

¹ Present address: Department of Epidemiology, University of Pittsburgh, Pittsburgh, PA 15261.

BAKER, B. S., A. T. C. CARPENTER and P. RIPOLL, 1978 The utilization during mitotic cell division of loci controlling meiotic recombination in Drosophila melanogaster. Genetics 90: 531–578.[Abstract/Free Full Text]

BAKER, B. S., M. GATTI, A. T. C. CARPENTER, S. PIMPINELLI and D. A. SMITH, 1980 Effects of recombination-deficient and repair-deficient loci on meiotic and mitotic chromosome behavior in Drosophila melanogaster, pp. 189–208 in DNA Repair and Mutagenesis in Eukaryotes, edited by W. M. GENEROSO, M. D. SHELBY and F. J. DE SERRES. Plenum Press, New York.

BAKER, N. E., 2001 Cell proliferation, survival, and death in the Drosophila eye. Semin. Cell Dev. Biol. 12: 499–507.[CrossRef][Medline]

BANGA, S. S., R. SHENKAR and J. B. BOYD, 1986 Hypersensitivity of Drosophila mei-41 mutants to hydroxyurea is associated with reduced mitotic chromosome stability. Mutat. Res. 163: 157–165.[CrossRef][Medline]

BHAUMIK, D., and T. S. WANG, 1998 Mutational effect of fission yeast polalpha on cell cycle events. Mol. Biol. Cell 9: 2107–2123.[Abstract/Free Full Text]

BI, X., M. GONG, D. SRIKANTA and Y. S. RONG, 2005 Drosophila ATM and Mre11 are essential for the G2/M checkpoint induced by low-dose irradiation. Genetics 171: 845–847.[Abstract/Free Full Text]

BISHOP, A. J., C. BARLOW, A. J. WYNSHAW-BORIS and R. H. SCHIESTL, 2000 Atm deficiency causes an increased frequency of intrachromosomal homologous recombination in mice. Cancer Res. 60: 395–399.[Abstract/Free Full Text]

BODDY, M. N., B. FURNARI, O. MONDESERT and P. RUSSELL, 1998 Replication checkpoint enforced by kinases Cds1 and Chk1. Science 280: 909–912.[Abstract/Free Full Text]

BONINI, N. M., and M. E. FORTINI, 1999 Surviving Drosophila eye development: integrating cell death with differentiation during formation of a neural structure. BioEssays 21: 991–1003.[CrossRef][Medline]

BOYD, J. B., M. D. GOLINO, T. D. NGUYEN and M. M. GREEN, 1976 Isolation and characterization of X-linked mutants of Drosophila melanogaster which are sensitive to mutagens. Genetics 84: 485–506.[Abstract/Free Full Text]

BRODSKY, M. H., J. SEKELSKY, G. TSANG, R. S. HAWLEY and G. M. RUBIN, 2000 mus304 encodes a novel DNA damage checkpoint protein required during Drosophila development. Genes Dev. 14: 666–678.[Abstract/Free Full Text]

BRODSKY, M. H., B. T. WEINERT, G. TSANG, Y. S. RONG, N. M. MCGINNIS et al., 2004 Drosophila melanogaster MNK/Chk2 and p53 regulate multiple DNA repair and apoptotic pathways following DNA damage. Mol. Cell. Biol. 24: 1219–1231.[Abstract/Free Full Text]

BROWN, E. J., and D. BALTIMORE, 2003 Essential and dispensable roles of ATR in cell cycle arrest and genome maintenance. Genes Dev. 17: 615–628.[Abstract/Free Full Text]

BYUN, T. S., M. PACEK, M. C. YEE, J. C. WALTER and K. A. CIMPRICH, 2005 Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint. Genes Dev. 19: 1040–1052.[Abstract/Free Full Text]

CANMAN, C. E., D.-S. LIN, K. A. CIMPRICH, Y. TAYA, K. TAMAI et al., 1998 Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science 281: 1677–1679.[Abstract/Free Full Text]

CASPER, A. M., P. NGHIEM, M. F. ARLT and T. W. GLOVER, 2002 ATR regulates fragile site stability. Cell 111: 779–789.[CrossRef][Medline]

CHA, R. S., and N. KLECKNER, 2002 ATR homolog Mec1 promotes fork progression, thus averting breaks in replication slow zones. Science 297: 602–606.[Abstract/Free Full Text]

CHEN, X., Q. LI and J. A. FISCHER, 2000 Genetic analysis of the Drosophila DNAprim gene: the function of the 60-kD primase subunit of DNA polymerase opposes the fat facets signaling pathway in the developing eye. Genetics 156: 1787–1795.[Abstract/Free Full Text]

CHEN, Y., and Y. SANCHEZ, 2004 Chk1 in the DNA damage response: conserved roles from yeasts to mammals. DNA Rep. 3: 1025–1032.[CrossRef]

CLIBY, W. A., C. J. ROBERTS, K. A. CIMPRICH, C. M. STRINGER, J. R. LAMB et al., 1998 Overexpression of a kinase-inactive ATR protein causes sensitivity to DNA-damaging agents and defects in cell cycle checkpoints. EMBO J. 17: 159–169.[CrossRef][Medline]

COBB, J. A., L. BJERGBAEK, K. SHIMADA, C. FREI and S. M. GASSER, 2003 DNA polymerase stabilization at stalled replication forks requires Mec1 and the RecQ helicase Sgs1. EMBO J. 22: 4325–4336.[CrossRef][Medline]

COBB, J. A., T. SCHLEKER, V. ROJAS, L. BJERGBAEK, J. A. TERCERO et al., 2005 Replisome instability, fork collapse, and gross chromosomal rearrangements arise synergistically from Mec1 kinase and RecQ helicase mutations. Genes Dev. 19: 3055–3069.[Abstract/Free Full Text]

CORTEZ, D., 2005 Unwind and slow down: checkpoint activation by helicase and polymerase uncoupling. Genes Dev. 19: 1007–1012.[Free Full Text]

CREST, J., N. OXNARD, J. Y. JI and G. SCHUBIGER, 2007 Onset of the DNA replication checkpoint in the early Drosophila embryo. Genetics 175: 567–584.[Abstract/Free Full Text]

DAS, K. C., and R. DASHNAMOORTHY, 2004 Hyperoxia activates the ATR-Chk1 pathway and phosphorylates p53 at multiple sites. Am. J. Physiol. Lung Cell. Mol. Physiol. 286: L87–L97.[Abstract/Free Full Text]

DE VRIES, H. I., L. UYETAKE, W. LEMSTRA, J. F. BRUNSTING, T. T. SU et al., 2005 Grp/DChk1 is required for G2-M checkpoint activation in Drosophila S2 cells, whereas Dmnk/DChk2 is dispensable. J. Cell Sci. 118: 1833–1842.[Abstract/Free Full Text]

D'URSO, G., B. GRALLERT and P. NURSE, 1995 DNA polymerase alpha, a component of the replication initiation complex, is essential for the checkpoint coupling S phase to mitosis in fission yeast. J. Cell Sci. 108(Pt. 9): 3109–3118.

EDGAR, B. A., F. SPRENGER, R. J. DURONIO, P. LEOPOLD and P. H. O'FARRELL, 1994 Distinct molecular mechanisms regulate cell cycle timing at successive stages of Drosophila embryogenesis. Genes Dev. 8: 440–452.[Abstract/Free Full Text]

FOIANI, M., G. LUCCHINI and P. PLEVANI, 1997 The DNA polymerase alpha-primase complex couples DNA replication, cell-cycle progression and DNA-damage response. Trends Biochem. Sci. 22: 424–427.[CrossRef][Medline]

FROSINA, G., 2000 Overexpression of enzymes that repair endogenous damage to DNA. Eur. J. Biochem. 267: 2135–2149.[Medline]

GARNER, M., S. VAN KREEVELD and T. T. SU, 2001 mei-41 and bub1 block mitosis at two distinct steps in response to incomplete DNA replication in Drosophila embryos. Curr. Biol. 11: 1595–1599.[CrossRef][Medline]

GATTI, M., 1979 Genetic control of chromosome breakage and rejoining in Drosophila melanogaster: spontaneous chromosome aberrations in X-linked mutants defective in DNA metabolism. Proc. Natl. Acad. Sci. USA 76: 1377–1381.[Abstract/Free Full Text]

GIRALDEZ, A. J., and S. M. COHEN, 2003 Wingless and Notch signaling provide cell survival cues and control cell proliferation during wing development. Development 130: 6533–6543.[Abstract/Free Full Text]

HARI, K. L., A. SANTERRE, J. SEKELSKY, K. S. MCKIM, J. B. BOYD et al., 1995 The mei-41 gene of D. melanogaster is a structural and functional homolog of the human ataxia telangiectasia gene. Cell 82: 815–821.

HAYNIE, J. L., and P. J. BRYANT, 1977 The effects of X-rays on the proliferation dynamics of cells in the imaginal wing disc of Drosophila mlelanogaster. Rouxs Arch. Dev. Biol. 183: 85–100.[CrossRef]

HELT, C. E., W. A. CLIBY, P. C. KENG, R. A. BAMBARA and M. A. O'REILLY, 2005 Ataxia telangiectasia mutated (ATM) and ATM and Rad3-related protein exhibit selective target specificities in response to different forms of DNA damage. J. Biol. Chem. 280: 1186–1192.[Abstract/Free Full Text]

HENDERSON, D. S., U. K. WIEGAND, D. G. NORMAN and D. M. GLOVER, 2000 Mutual correction of faulty PCNA subunits in temperature-sensitive lethal mus209 mutants of Drosophila melanogaster. Genetics 154: 1721–1733.[Abstract/Free Full Text]

JACOBS, H. W., J. A. KNOBLICH and C. F. LEHNER, 1998 Drosophila Cyclin B3 is required for female fertility and is dispensable for mitosis like cyclin B. Genes Dev. 12: 3741–3751.[Abstract/Free Full Text]

JAKLEVIC, B. R., and T. T. SU, 2004 Relative contribution of DNA repair, cell cycle checkpoints, and cell death to survival after DNA damage in Drosophila larvae. Curr. Biol. 14: 23–32.[CrossRef][Medline]

JEGGO, P. A., and M. LOBRICH, 2006 Contribution of DNA repair and cell cycle checkpoint arrest to the maintenance of genomic stability. DNA Rep. 5: 1192–1198.[CrossRef]

JEGGO, P. A., A. M. CARR and A. R. LEHMANN, 1998 Splitting the ATM: distinct repair and checkpoint defects in ataxia-telangiectasia. Trends Genet. 14: 312–316.[CrossRef][Medline]

LAROCQUE, J. R., B. JACKLEVIC, T. T. SU and J. SEKELSKY, 2007 Drosophila ATR in double-strand break repair. Genetics 175: 1023–1033.[Abstract/Free Full Text]

LAURENCON, A., A. PURDY, J. SEKELSKY, R. S. HAWLEY and T. T. SU, 2003 Phenotypic analysis of separation-of-function alleles of MEI-41, Drosophila ATM/ATR. Genetics 164: 589–601.[Abstract/Free Full Text]

LEE, J. H., E. LEE, J. PARK, E. KIM, J. KIM et al., 2003 In vivo p53 function is indispensable for DNA damage-induced apoptotic signaling in Drosophila. FEBS Lett. 550: 5–10.[CrossRef][Medline]

LEMOINE, F. J., N. P. DEGTYAREVA, K. LOBACHEV and T. D. PETES, 2005 Chromosomal translocations in yeast induced by low levels of DNA polymerase: a model for chromosome fragile sites. Cell 120: 587–598.[CrossRef][Medline]

LINDAHL, T., 1993 Instability and decay of the primary structure of DNA. Nature 362: 709–715.[CrossRef][Medline]

LIU, Q., S. GUNTUKU, X. S. CUI, S. MATSUOKA, D. CORTEZ et al., 2000 Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. Genes Dev. 14: 1448–1459.[Abstract/Free Full Text]

LOBRICH, M., and P. A. JEGGO, 2005 Harmonising the response to DSBs: a new string in the ATM bow. DNA Rep. 4: 749–759.[CrossRef]

MASROUHA, N., L. YANG, S. HIJAL, S. LAROCHELLE and B. SUTER, 2003 The Drosophila chk2 gene loki is essential for embryonic DNA double-strand-break checkpoints induced in S phase or G2. Genetics 163: 973–982.[Abstract/Free Full Text]

O'DRISCOLL, M., and P. A. JEGGO, 2006 The role of double-strand break repair: insights from human genetics. Nat. Rev. Genet. 7: 45–54.[CrossRef][Medline]

O'DRISCOLL, M., A. R. GENNERY, J. SEIDEL, P. CONCANNON and P. A. JEGGO, 2004 An overview of three new disorders associated with genetic instability: LIG4 syndrome, RS-SCID and ATR-Seckel syndrome. DNA Rep. 3: 1227–1235.[CrossRef]

OIKEMUS, S. R., J. QUEIROZ-MACHADO, K. LAI, N. MCGINNIS, C. SUNKEL et al., 2006 Epigenetic telomere protection by Drosophila DNA damage response pathways. PLoS Genet. 2: e71.

OLLMANN, M., L. M. YOUNG, C. J. DI COMO, F. KARIM, M. BELVIN et al., 2000 Drosophila p53 is a structural and functional homolog of the tumor suppressor p53. Cell 101: 91–101.[CrossRef][Medline]

PâQUES, F., and J. E. HABER, 1999 Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 63: 349–404.[Abstract/Free Full Text]

RONG, Y. S., S. W. TITEN, H. B. XIE, M. M. GOLIC, M. BASTIANI et al., 2002 Targeted mutagenesis by homologous recombination in D. melanogaster. Genes Dev. 16: 1568–1581.[Abstract/Free Full Text]

ROYOU, A., H. MACIAS and W. SULLIVAN, 2005 The Drosophila Grp/Chk1 DNA damage checkpoint controls entry into anaphase. Curr. Biol. 15: 334–339.[CrossRef][Medline]

ROYZMAN, I., A. J. WHITTAKER and T. L. ORR-WEAVER, 1997 Mutations in Drosophila DP and E2F distinguish G1-S progression from an associated transcriptional program. Genes Dev. 11: 1999–2011.[Abstract/Free Full Text]

SANCAR, A., L. A. LINDSEY-BOLTZ, K. UNSAL-KACMAZ and S. LINN, 2004 Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu. Rev. Biochem. 73: 39–85.[CrossRef][Medline]

SANCHEZ, Y., B. A. DESANY, W. J. JONES, Q. LIU, B. WANG et al., 1996 Regulation of RAD53 by the ATM-like kinases MEC1 and TEL1 in yeast cell cycle checkpoint pathways. Science 271: 357–360.[Abstract]

SHILOH, Y., 2003 ATM: ready, set, go. Cell Cycle 2: 116–117.[Medline]

SIBON, O. C., A. LAURENCON, R. HAWLEY and W. E. THEURKAUF, 1999 The Drosophila ATM homologue Mei-41 has an essential checkpoint function at the midblastula transition. Curr. Biol. 9: 302–312.[CrossRef][Medline]

SIGRIST, S. J., and C. F. LEHNER, 1997 Drosophila fizzy-related down-regulates mitotic cyclins and is required for cell proliferation arrest and entry into endocycles. Cell 90: 671–681.[CrossRef][Medline]

SILVA, E., S. TIONG, M. PEDERSEN, E. HOMOLA, A. ROYOU et al., 2004 ATM is required for telomere maintenance and chromosome stability during Drosophila development. Curr. Biol. 14: 1341–1347.[CrossRef][Medline]

SU, T. T., S. D. CAMPBELL and P. H. O'FARRELL, 1999 Drosophila grapes/CHK1 mutants are defective in cyclin proteolysis and coordination of mitotic events. Curr. Biol. 9: 919–922.[CrossRef][Medline]

UNSAL-KACMAZ, K., A. M. MAKHOV, J. D. GRIFFITH and A. SANCAR, 2002 Preferential binding of ATR protein to UV-damaged DNA. Proc. Natl. Acad. Sci. USA 99: 6673–6678.[Abstract/Free Full Text]

VAN LANCKER, J. L., 2006 Apoptosis, Genome Integrity, and Cancer. Jones & Bartlett, Sudbury, MA.

WALTER, J., and J. NEWPORT, 2000 Initiation of eukaryotic DNA replication: origin unwinding and sequential chromatin association of Cdc45, RPA, and DNA polymerase alpha. Mol. Cell 5: 617–627.[CrossRef][Medline]

WRIGHT, J. A., K. S. KEEGAN, D. R. HERENDEEN, N. J. BENTLEY, A. M. CARR et al., 1998 Protein kinase mutants of human ATR increase sensitivity to UV and ionizing radiation and abrogate cell cycle checkpoint control. Proc. Natl. Acad. Sci. USA 95: 7445–7450.[Abstract/Free Full Text]

XU, J., S. XIN and W. DU, 2001 Drosophila Chk2 is required for DNA damage-mediated cell cycle arrest and apoptosis. FEBS Lett. 508: 394–398.[CrossRef][Medline]

YU, K. R., R. B. SAINT and W. SULLIVAN, 2000 The Grapes checkpoint coordinates nuclear envelope breakdown and chromosome condensation. Nat. Cell Biol. 2: 609–615.[CrossRef][Medline]

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