ABSTRACT
The RAD54 gene has an essential role in the repair of double-strand breaks (DSBs) via homologous recombination in yeast as well as in higher eukaryotes. A Drosophila melanogasterstrain deficient in the RAD54 homolog DmRAD54is characterized by increased X-ray and methyl methanesulfonate (MMS) sensitivity. In addition, DmRAD54 is involved in the repair of DNA interstrand cross-links, as is shown here. However, whereas X-ray-induced loss-of-heterozygosity (LOH) events were completely absent in DmRAD54−/− flies, treatment with cross-linking agents or MMS resulted in only a slight reduction in LOH events in comparison with those in wild-type flies. To investigate the relative contributions of recombinational repair and nonhomologous end joining in DSB repair, aDmRad54 −/−/DmKu70 −/−double mutant was generated. Compared with both single mutants, a strong synergistic increase in X-ray sensitivity was observed in the double mutant. No similar increase in sensitivity was seen after treatment with MMS. Apparently, the two DSB repair pathways overlap much less in the repair of MMS-induced lesions than in that of X-ray-induced lesions. Excision of P transposable elements inDrosophila involves the formation of site-specific DSBs. In the absence of the DmRAD54 gene product, no male flies could be recovered after the excision of a single P element and the survival of females was reduced to 10% compared to that of wild-type flies. P-element excision involves the formation of two DSBs which have identical 3′ overhangs of 17 nucleotides. The crucial role of homologous recombination in the repair of these DSBs may be related to the very specific nature of the breaks.
Double-strand breaks (DSBs) are important DNA lesions that, if left unrepaired, can lead to broken chromosomes and cell death and, if repaired improperly, can result in chromosomal rearrangements. In cells of higher organisms, the formation and repair of DSBs are intrinsic to a number of biological processes such as meiotic recombination, V(D)J recombination in differentiating lymphocytes, and transposition of certain mobile DNA elements. Furthermore, DSBs can be induced upon exposure to exogeneous factors such as ionizing radiation and may arise indirectly after treatment with chemical agents such as methyl methanesulfonate (MMS) and cross-linking agents. In eukaryotes, repair of DSBs can occur by at least two pathways: (i) nonhomologous end joining (NHEJ) and (ii) repair via homologous recombination (recombinational repair).
Repair of DSBs by NHEJ was first studied in higher eukaryotes by using rodent cell mutants (11, 44). Genes known to be involved in this pathway are DNA-PKcs, Ku80, Ku70,XRCC4, and Ligase IV. Cell lines with defects in NHEJ are impaired in V(D)J rearrangement and are sensitive to ionizing radiation. Homologs of Ku70, Ku80,XRCC4, and Ligase IV have been identified inSaccharomyces cerevisiae (6, 16, 23, 35, 43, 45). Yeast mutants defective in one of these genes are hardly sensitive to ionizing radiation. A contribution of NHEJ can be detected only in the absence of recombinational repair, indicating that recombinational repair is the predominant mechanism for DSB repair in yeast (7, 28, 38). The homolog of Ku70 in Drosophila melanogaster, DmKu70, is encoded by the mus309 gene (1, 2, 24). Flies with mutations of this gene are hypersensitive to MMS (9).
Repair of DSBs through homologous recombination has been extensively studied in the yeast S. cerevisiae and is dependent on the RAD52 group genes (RAD50, RAD51,RAD52, RAD54, RAD55, RAD57,RAD59, MRE11 [RAD58/XRS4],XRS2, and RDH54/TID1) (references25 and 37; reviewed in references18 and 30). Yeast strains deficient in one of these genes have an increased sensitivity to ionizing radiation; rad51, rad52, andrad54 mutants have the most severe phenotype. Furthermore, these three mutants have defects in spontaneous and induced mitotic recombination and mating type switching, rad51 andrad52 mutants are also impaired in meiotic recombination, and the formation of viable spores is almost completely blocked. Rad51 is the eukaryotic homolog of RecA and has limited pairing and strand exchange activities (39). The strand exchange activities of Rad51 are stimulated by Rad52 or by a heterodimer of Rad55 and Rad57 by overcoming the inhibitory effects of replication protein A (29, 36, 40, 41). The Rad54 protein, which has a double-stranded DNA-dependent ATPase activity, also stimulates the Rad51-dependent formation of heteroduplex DNA (33).
During the past few years, homologs of the RAD52 group genes have been identified in higher eukaryotes. Both structurally and functionally these homologs resemble their counterparts from yeast (18, 32). Recently, we have identified the RAD54homolog in D. melanogaster, DmRAD54(26). Flies with mutations in this gene are characterized by an increased sensitivity of the larvae to X rays and MMS. Furthermore, the DmRAD54 mutant is defective in X-ray-induced recombination in somatic cells. Similar results have been reported forRAD54-deficient mouse embryonic stem cells and chicken DT40 cells (5, 15). In contrast toRAD54 −/− mice, DmRAD54-deficient flies have meiotic defects. Mutant males are fertile, but females are sterile due to inviability of the eggs. Defects in meiotic recombination, which occurs only in females in Drosophila, possibly affects patterning during oogenesis, as was recently shown by Ghabrial et al. (19). Together, the analysis ofRAD54-deficient fly strains and mouse and chicken cells demonstrates that, besides NHEJ, recombinational repair contributes to the repair of DSBs in higher eukaryotes.
In the present study we have examined the role ofDmRAD54 in DSB repair in more detail. One important source of site-specific DSBs in Drosophilais the excision of P transposable elements. Full-length P elements are 2.9 kb in length and contain 31-bp terminal inverted repeats (31). The P-element-encoded transposase binds to inverted repeats and introduces two site-specific DSBs with identical 17-nucleotide 3′ extensions (3). Genetic studies have shown that repair of the breaks can occur by gene conversion using the sister chromatid, the homologous chromosome, or an ectopically located homologous sequence as a template, indicating an important role for the recombinational-repair mechanism (14). To study the role of DmRAD54 in this process, we introduced aDmRAD54 mutation in flies containing all the elements required for P-element excision. The results obtained indicate an important role for DmRad54 in the repair of DSBs after P-element excision.
The involvement of recombinational repair in overcoming cross-links in the DNA was examined by determining the larval sensitivity ofDmRAD54 mutant flies to the cross-linking agents mitomycin C (MMC) and cis-diamminedichloroplatinum (cisDDP). Simultaneously, we examined the levels of loss of heterozygosity (LOH) in somatic cells after treatment with these agents or MMS. To investigate the relative contributions of NHEJ and recombinational repair in Drosophila, a DmRAD54mutation was combined with a mutation in the DmKu70 gene. Survival experiments suggest a strong synergism between recombinational repair and NHEJ in the repair of X-ray damage but not in the repair of MMS-induced DNA damage.
MATERIALS AND METHODS
Drosophila strains and chromosomes.TheDmRAD54-deficient fly strain DmRAD54A17-11cn bw/Df(2L)JS17 has been described previously (26). Briefly, the A17-11 mutation is a GC-to-AT transition at the splice acceptor site of the second intron in the DmRAD54gene. The DmRAD54A17-11 allele is referred to below as A17-11, and deficiency chromosomeDf(2L)JS17 is referred to as JS17. From the experiment that resulted in the isolation of the A17-11mutant, a second DmRAD54 mutant, calledDmRAD54A19-10 cn bw (referred to below asA19-10), was isolated. This mutant contains an AT-to-GC transition mutation resulting in a V146D amino acid alteration. The valine at this position is also present in Rad54 proteins from yeast and mammals. Hemizygous mutants A19-10/JS17 and heteroallelic homozygous A19-10/A17-11 mutants have the same MMS and X-ray hypersensitivity as the A17-11/JS17 mutant strain, and the females in both mutant strains are sterile.
For the construction of a DmRAD54/mus309(DmKu70) double mutant, balancer chromosomes with visible dominant marker mutations were used; Cy and Pm indicate the second chromosome balancers SM5 and In(2LR)bwV1 , respectively, and Ubx and Sb indicate the third chromosome balancers TM2 and TM3, respectively (27). These chromosomes suppress meiotic recombination and can be monitored directly in the F1 generation due to the presence of the visible dominant markers. The DmRAD54 −/−mutant was made by combining the allele A17-11 with the allele A19-10. JS17 was not used for the construction of the double mutant, since strains with this deletion combined with amus309 allele and two balancer chromosomes showed very poor viability. Two alleles, mus309D2 andmus309D3 , had to be combined to obtain a homozygous mus309 mutant, since both chromosomes contain additional recessive lethal mutations. The final cross that was made to determine the sensitivities of the single mutants and the double mutant to ionizing radiation was A17-11 cn bw/Cy; mus309D2/Ubx × A19-10 cn bw/Pm; mus309D3/Sb and resulted in 16 different phenotypes in the F1 offspring. According to Mendelian laws, nine types of flies are homozygous or heterozygous wild type for both genes, three types are homozygous mus309 mutants, three types are homozygous DmRAD54 mutants, and only one type is a double mutant.
The somatic mutation and recombination test (SMART) was performed as described previously (26). For each point in the assay, at least 200 eyes were scored.
Drosophila strains y wa spl andy whd spl; P[walter ]25F/CyO were kindly provided by Carlos Flores and William R. Engels. Thewa allele results from a copia- element insertion in the second intron of the white gene. Themus309 alleles D2 and D3 as well as the Df(2L)JS17 strain, were kindly provided by Jeff Sekelsky. Other strains were obtained from the DrosophilaStock Center in Bloomington, Ind., and are described by Lindsley and Zimm (27) and in FlyBase (17).
P-element excision assay.To study DSB repair after P-element excision, the whd (whd80617 ) element was used. This mutant allele of the X-chromosome-linked white gene carries a small P element of 629 bp in exon 6 (20). P[walter ] was incorporated in the system as a template for recombination. The P[walter ] element contains an altered white minigene in a P-element vector located in region 25F of the second chromosome (20). Although the white minigene carries 12 base pair substitutions, it encodes a wild-type white gene product. Due to position effects at the site of integration at 25F, thewalter gene is hardly expressed. By standard genetic crosses a recombinant second chromosome was obtained, carrying the JS17 deletion, which uncovers the DmRAD54gene together with walter . The transposase required for excision of the whd element is encoded by the P[ry +Δ2-3] element (abbreviated as Δ2-3) at position 99B on the third chromosome (24). To combine all the elements required for P-element excision and repair, y wa spl; A17-11 cn bw/Cy; Sb P[ry +Δ2-3]/Ubxmales were crossed to whd/whd; JS17 walter/Cy females. In theDmRAD54-proficient control cross,whd/whd; walter/Cyfemales were used (see Fig. 1).
Treatment of Drosophila with DNA-damaging agents.Sensitivity to different DNA-damaging agents is dependent on the developmental stage of Drosophila, and therefore larvae of different stages were used for the various treatments. To obtain optimal results, 0- to 24-h embryos were X-ray irradiated, 24- to 48-h larvae were treated with 0.2 ml of MMS in water, and 48- to 72-h larvae were treated with 0.2 ml of MMC in water or 0.2 ml of cis-DDP in 0.4% dimethylformamide. Untreated larvae of the same parents were used as controls. Unless noted otherwise,A17-11/Cy females were crossed to JS17/Cy males. Fly cultures were grown at 25°C, and the offspring was analyzed after 12 to 18 days. To calculate the relative sensitivity, the ratio between homozygous mutants A17-11/JS17 and heterozygousA17-11/Cy and JS17/Cy flies was determined. This ratio is theoretically 0.5 in untreated samples, according to Mendelian laws. If the sensitivity to DNA-damaging agents is increased, this ratio of 0.5 will decrease as the dose increases. The relative sensitivity can be calculated by dividing the ratio of the untreated sample by the ratio of the treated sample. In the case of theDmRAD54/mus309 double mutant, the ratio between homozygous or heterozygous wild-type flies, mus309 mutants,DmRAD54 mutants, and double-mutant flies is 9:3:3:1 according to Mendelian laws. After treatment the number of flies of each class is corrected for these ratios before the relative sensitivities are calculated.
RESULTS
DmRAD54 is crucial for DSB repair after P-element transposition.One of the first steps in P-element transposition in Drosophila is the formation of two staggered DSBs at the P-element termini. Reversion of the donor site to wild type is dependent on the presence of sequence information on the homologous chromosome or an ectopic template. To investigate the role ofDmRAD54 in this process, DSB repair after P-element excision was studied in a DmRAD54 −/− background. Thewhd allele of white, which carries a small P-element insertion in exon 6, was used as a detection system for correct repair. Flies homozygous for the whd mutation have bleached white eyes. The walter sequence on the second chromosome can be used as a template for recombinational repair. The transposase required for excision of thewhd element is supplied by crossingwhd females to male flies carrying the Δ2-3 element on the third chromosome, which provides the transposase protein for induction of site-specific DSBs (34). A second chromosome, carrying only thewalter element and not the JS17deletion, was used as a DmRAD54-proficient control. Figure1B shows the cross that was used and the two types of flies (male and female) that emerge from this cross carrying all the necessary components. According to Mendelian laws, in the offspring of theDmRAD54-deficient as well as theDmRAD54-proficient cross, half of the flies that are recovered carry the transposase source (marked by the dominantSb mutation). Excision of whd by the transposase during development may give rise to cells expressing a wild-type white gene as a result of recombinational repair using walter as a template. Clonal expansion results in spots of wild-type colored tissues against a colorless background in the eyes of the adult flies. Male flies from the initial cross (Fig. 1B) carried the mutant whiteapricot (wa ) allele. As a result the female offspring also have white eyes. In females the wa allele can also be used as a template for homologous recombination in addition to walter . Table 1shows the numbers of the most indicative flies that emerged from the cross and the numbers of eyes from these flies that contained one or more spots. In the DmRAD54-proficient control, the different groups of flies hatched in equal ratios. Flies without the transposase (indicated by the Ubx marker) did not have red spots, as expected, and a majority of the eyes in flies with the transposase contained one or more red spots. However, from theDmRAD54-deficient cross, only flies without the transposase emerged in a normal ratio. MaleDmRAD54 −/− larvae in which P-element excision was induced did not survive at all, and the proportion of female DmRAD54 −/− flies was reduced to 10% relative to the females that did not have the transposase. Furthermore, the frequency of red spots in theDmRAD54 −/− female flies that did emerge was reduced fivefold compared to that in the correspondingDmRAD54 +/− female group. These results clearly show a crucial role for DmRAD54 in the repair of P-element transposase-induced DSBs.
(A) Mechanism of induction and repair of a single DSB after P-element excision. The whd element has an internal deletion and does not code for a functional P-element transposase. It is excised by the transposase encoded by the Δ2-3 element on the third chromosome, leaving behind a DSB. In males this DSB can be repaired via homologous recombination using the walter template, located at position 25F on the second chromosome, resulting in a functionalwhite gene. Repair via NHEJ will in most cases result in a nonfunctional white gene. Females can also use thewa allele on the other X chromosome as a template. (B) Cross to combine the whd transposition system with a DmRAD54-deficient background. Cy and Ubx are dominant marker mutations on the second and third balancer chromosomes, respectively. The third chromosome, carrying the Δ2-3 element, is marked by the dominant Sb mutation. The males used in this cross carry the wa mutation on the X chromosome to ensure a white-eye background in the female offspring. In the control cross, DmRAD54-proficient females carrying a second chromosome containing only the walter construct were used.
Survival and reversion frequency after P-element excision
DmRAD54 is involved in the repair of DNA cross-links.Recently, we showed that DmRAD54 is involved in the repair of DNA damage induced by ionizing radiation and MMS. To determine if homologous recombination is involved in cross-link repair, the sensitivity of the DmRAD54−/−mutant to MMC and cisDDP was tested. HeterozygousDmRAD54 +/− flies were crossed, and the offspring were treated as 48- to 72-h-old larvae with increasing doses of MMC or cisDDP. The relative sensitivities were calculated using the ratios of emerging DmRAD54 −/− flies andDmRAD54 +/− flies with or without treatment as described in Materials and Methods. Table2 shows a dose-dependent increase in sensitivity to both agents for theDmRAD54 −/− flies. At a dose of 0.3 mM MMC, the sensitivity was increased by a factor of 6.8. In the case of treatment with 1 mM cisDDP, the DmRAD54 −/−larvae were 6.4 times more sensitive than theDmRAD54 +/− control larvae. After exposure to higher doses, or at an earlier stage of development, the sensitivities increased further, but these doses were too toxic for the control group and sensitivities could not be calculated accurately any more.
Sensitivity to cross-linking agents
Somatic recombination induced by cross-linking agents or MMS occurs at a reduced level in DmRAD54-deficient flies.It has previously been shown that somatic recombination induced by X rays was completely absent in DmRAD54 −/− flies (26). This system measures the LOH of both wild-type genes for cinnabar (cn) and brown(bw) in a heterozygous (cn bw/++) larval eye precursor cell due to a mutagenic event. Clonal expansion results in a colorless spot against a wild-type red background in the eyes of adult flies. The sensitivity to the alkylating agent MMS and to the cross-linking agents MMC and cisDDP raised the question whether the induction of LOH events by these agents is also affected. Figure 2 shows that treatment with each mutagen resulted in a dose-dependent increase in the number of spots per eye in DmRAD54-proficient flies. In theDmRAD54 −/− flies, no induction of spots was seen after exposure to ionizing radiation (Fig. 2A). However, after exposure to MMS, MMC, or cisDDP, induction of spots is still possible in DmRAD54 mutant flies, although at a reduced level compared to that in the control group (Fig. 2B through D). Apparently, the majority of LOH events induced by these chemical agents occur in aDmRAD54-independent way.
Somatic recombination induced by DNA-damaging agents in a DmRAD54-deficient background.DmRAD54-proficient control larvae (▵) andDmRAD54-deficient larvae (▴) were treated with increasing doses of ionizing radiation (A), the cross-linking agent MMC (B) or cisDDP (C), or the alkylating agent MMS (D). Twelve to 18 days after treatment, the eyes of the adult flies were scored for colorless spots. Each spot is caused by one LOH event. Results are presented as spots per eye; for each point, at least 200 eyes were scored.
DmRAD54 and DmKu70 act synergistically in the repair of X-ray damage but not in the repair of damage induced by MMS.To examine the relative contributions of recombinational repair and NHEJ in Drosophila, a double mutant was generated. For this purposeDmRAD54 +/−/DmKu70 +/−heterozygous females and males were crossed, and the larvae obtained were treated with X rays or MMS. From this cross double-mutant flies, both single mutants, and homozygous or heterozygous wild-type control flies were obtained, allowing a direct comparison of the sensitivities of the various mutants to DNA-damaging agents. Table3 shows the numbers of each type after mock treatment or treatment with 6 Gy of X rays or 0.06 mM MMS. The numbers of the flies recovered were normalized for the Mendelian ratios and used to calculate the relative sensitivity of each group. In the untreated sample, the ratio of 9:4:3.4:1.2 deviates only slightly from the expected ratio of 9:3:3:1. After irradiation, a decrease in the ratios was observed for both single mutants and a very strong decrease was observed for the double mutant. At a dose of 6 Gy of X rays, theDmKu70 mutant flies showed threefold-enhanced sensitivity and the DmRAD54 mutant flies showed sevenfold-enhanced sensitivity. The radiation sensitivity of theDmRAD54/DmKu70 double mutant was increased 40-fold (see Table 3). Statistical analysis showed that the 95% confidence interval for this increase in sensitivity has a minimum of a factor of 17. Therefore, the conclusion is that DmRAD54and Ku70 mutations cause a clear synergistic effect. This synergism in X-ray sensitivity indicates that theDmRAD54 and DmKu70 genes are involved in separate DSB repair pathways and that X-ray-inflicted damage can be repaired by both recombinational repair and NHEJ. However, a synergistic effect was not observed after treatment of the double mutant with MMS: the DmKu70 single mutant was hardly sensitive to 0.06 mM MMS (a twofold increase), the sensitivity of theDmRAD54 single mutant was increased eightfold, and an intermediate increase in sensitivity (sixfold) was observed in theDmRAD54/DmKu70 double mutant. The 95% confidence interval for the sensitivity of the double mutant to MMS is between a minimum of 3-fold and a maximum of 12-fold, indicating that the effect could be additive but is not synergistic.
Sensitivity of aDmRAD54 −/−/DmKu70 −/−double mutant to X rays and MMS
DISCUSSION
Homologous recombination is the major pathway for the repair of DSBs in yeast. The Rad54 protein, a member of the Snf2/Swi2 subfamily of DNA-dependent ATPases (21), is one of the main factors in this pathway. The analysis of mouse and chicken cells deficient inRAD54 and of a Drosophila strain with a mutation in DmRAD54 indicated that recombinational repair contributes significantly to the repair of DSBs in higher eukaryotes (5, 15, 26). A DmRAD54 null mutant shows increased larval sensitivity to ionizing radiation and MMS, defects in X-ray-induced recombination in somatic cells, and female sterility. During development the DmRAD54 gene is not essential, as evidenced by the fact that DmRAD54 −/−flies can be readily obtained after heterozygous males and females are crossed. It is possible, therefore, that maternally depositedDmRAD54 mRNA contributes to the repair of exogenously induced DNA damage during early development. The increase in sensitivity to DNA-damaging agents, therefore, could be underestimated.
In the past, the transposition mechanism of P transposable elements has been extensively studied in Drosophila. Recently, it has been shown that the first step in the transposition event involves the formation of two site-specific DSBs (3). Repair of these breaks can occur by recombination. To examine whetherDmRAD54 plays a part in this process, we determined the somatic reversion to wild type of whd inDmRAD54 +/− andDmRAD54 −/− flies. HeterozygousDmRAD54 larvae hatched as adults in a normal Mendelian ratio, with a reversion frequency of one or more red spots per eye in 70% of the male flies carrying the Δ2-3 transposase gene and 85% of the females (see Table 1). The females that emerged from both crosses contain the wa allele on the second X chromosome. This mutant allele of white may be used as a template for recombination in addition to thewalter element on the second chromosome. This could explain the increase in the frequency of spot-containing eyes in repair-proficient females in comparison with male flies. The effect of the DmRAD54 mutation was so great that male larvae in which P-element excision had occurred did not survive at all. The survival of female larvae was reduced to 10% in comparison with females from the same cross that did not have the transposase gene. In the flies that did hatch, the relative spot frequency was reduced fivefold compared to that in the control group. The difference in survival between males and females may be explained by the presence of the wa allele on the second X chromosome, which may give rise to a low level of DmRAD54-independent recombination in females. The results obtained in repair-deficient flies demonstrate that DmRAD54 is crucial for the repair of the DSBs after excision of a P element. Furthermore, the results suggest that NHEJ compensates only partially for the absence ofDmRAD54, or only in certain cell types. P-element excision has also been studied in DmKu70 mutant flies. With the X-linked snw system, the survival of males is reduced sixfold in a Ku70-deficient background (2). For the females, the reduction in survival was less than twofold. These data suggest that in the presence of a duplicate sequence, recombinational repair is the preferred mechanism. Most likely, due to the absence of a template for recombination, the effect was much stronger in the males used by Beall and Rio (2) than in the females. The conclusion that recombinational repair is the most important repair mechanism after P-element excision is supported by studies with a mus209 mutant which is deficient in proliferating cell nuclear antigen (PCNA) (22). In the absence of a functional PCNA product, mobilization of P elements results in lethality, presumably due to the essential role of PCNA in DNA repair synthesis during recombination. The prevalence of repair of DSBs by homologous recombination after P-element excision may be related to the specific nature of the breaks, which have identical 3′-single-stranded regions of 17 nucleotides. Possibly, repair of these breaks by NHEJ is not very efficient.
Cross-linking agents such as MMC and cisDDP produce a variety of DNA lesions, including cross-links. Repair of these lesions inEscherichia coli is dependent on both nucleotide excision repair (NER) and recombinational repair (12, 13). In mammalian cells the NER pathway is also involved in cross-link repair. Experiments of Bessho et al. using cell extracts or a reconstituted excision nuclease indicated that two incisions are made 22 to 28 nucleotides apart, both 5′ of the cross-link (4). InDrosophila the mei9 and mus201mutants, which are defective in the incision step in NER, also display larval sensitivity to the cross-linking agent nitrogen mustard (8, 10). To determine if the recombinational repair pathway also participates in cross-link repair in Drosophila, wild-type and DmRAD54-deficient larvae were treated with MMC and cisDDP. The sixfold increase in larval sensitivity to both agents (Table 2) at the highest doses used demonstrates that, as in E. coli and mammals (12, 13, 15), recombinational repair is also involved in the repair of cross-links in Drosophila.
LOH was studied by using a SMART. Loss of both cn andbw marker genes on the second chromosome results in regions of mutant tissue in the eyes of adult flies, which can be scored as colorless spots. Treatment with X rays, MMS, MMC, or cisDDP resulted in a dose-dependent increase in LOH events in wild-type flies (see Fig. 2). As previously described, no induction of spots was observed after X-ray treatment ofDmRAD54-deficient flies (26). Apparently, most of the X-ray-induced LOH events are due to aDmRAD54-dependent mechanism. In the absence ofDmRAD54, treatment with MMS, MMC, and cisDDP resulted in a dose-dependent induction of spots in the eyes, although the spot frequencies were reduced in comparison with those inDmRAD54-proficient flies. These results indicate that the chemical agents used induceDmRAD54-dependent and -independent events. TheDmRAD54-dependent events are probably due to homologous recombination, as has been previously described for ionizing radiation. DSBs are induced directly by ionizing radiation but only indirectly by MMS, MMC, and cisDDP as a consequence of repair and/or replication. It is feasible that these DSBs, or some of them, result inDmRAD54-independent LOH events.
To examine the relative contributions of homologous recombination and NHEJ in DSB repair, a DmRAD54/DmKu70 double mutant was generated. Both single mutants showed a slight increase in sensitivity to 6 Gy of X rays (seven- and threefold, respectively). The small difference in hypersensitivity for both single mutants could indicate that both mechanisms contribute more or less equally to the repair of X-ray-induced DSBs. These data, however, do not exclude the possibility that the relative contributions differ for the various cell types in the larvae. In the DmRAD54/mus309 double mutant, a clear synergistic effect was observed. At a dose of 6 Gy, the sensitivity was increased 40-fold. The small increase in radiation sensitivity seen in both single mutants in comparison with the double mutant strongly suggests overlap between recombinational repair and NHEJ at the larval stage of development. The significant increase in X-ray sensitivity in the double mutant suggests that repair by other mechanisms is not very efficient. In yeast, NHEJ is possible at a reduced level in the absence of Ku70 and involves the introduction of deletions at the site of the break (7). The same phenomenon has been observed in Drosophila by Beall and Rio (2). Another possibility is the presence of a second RAD54 homolog inDrosophila, as has been described in yeast (25, 36), which can substitute for DmRAD54.
In contrast to X rays, treatment with MMS did not result in a synergistic increase in sensitivity in the double mutant in comparison with both single mutants (see Table 3). This result differs from the results of survival experiments usingKu70 −/−/RAD54 −/−chicken DT40 cells, which showed a moderate sensitivity to ionizing radiation as well as to MMS in comparison with both single mutants (42). Exposure to MMS does not result in the direct induction of DSBs in DNA but possibly leads indirectly to the occurrence of DSBs as a consequence of repair or bypass of single-strand breaks during replication. Recent studies withKu70 −/−/RAD54 −/−chicken DT40 cells indicated that during the late S and G2phases of the cell cycle, DSBs are repaired predominantly by recombinational repair. In G1-phase cells, NHEJ is the prevalent mechanism (42). Therefore, preferential repair by homologous recombination during the S and G2 phases seems the most likely explanation for the absence of a strong synergistic increase in sensitivity in theKu70 −/−/DmRAD54 −/−flies after MMS treatment. With ionizing radiation, DSBs are induced at every stage of the cell cycle, leading to a drastic increase in sensitivity in the double mutant. An alternative explanation for the absence of a drastic increase in MMS sensitivity in the double mutant could be the formation of single-strand gaps as a result of repair or replicational bypass of MMS-induced lesions. These gaps may not be a substrate for the Ku-dependent pathway, resulting in comparable MMS sensitivities in the Ku70/DmRAD54 double mutant and theDmRAD54 single mutant.
In conclusion, the Drosophila DmRAD54 gene is involved in recombinational repair of DSBs, induced by the endogenous P-element transposase or exogenous factors such as ionizing radiation, MMS, and cross-linking agents, in addition to its function in meiotic cells. The synergistic increase in X-ray sensitivity in aKu70 −/−/DmRAD54 −/−double mutant and the absence of such an increase in sensitivity to MMS suggest that the stage of the cell cycle is an important factor in determining the relative contribution of each DSB repair pathway. Another important factor may be the nature of the DSB itself, as shown by the requirement for DmRAD54 in the repair of DSBs with identical 3′ overhangs, generated as a result of P-element excision.
ACKNOWLEDGMENTS
The work described in this paper was supported by the Dutch Cancer Foundation (project RUL94-774) and by the J. A. Cohen Institute, Interuniversity Research Institute for Radiopathology and Radiation Protection (IRS; project 4.4.12).
We thank Niels de Wind for critical reading of the manuscript and Koos Zwinderman for statistical analysis.
FOOTNOTES
- Received 22 April 1999.
- Returned for modification 23 May 1999.
- Accepted 14 June 1999.
- Copyright © 1999 American Society for Microbiology
