The influence of low doze gamma-irradiation on life span of Drosophila mutants with defects of DNA damage sensation and repair

It was shown that life span of wild type strain Canton-S increased after low doze gamma-irradiation. It was revealed the decrease of life span after irradiation in Drosophila mutants with defects of DNA damage sensation and repair genes mei-9 and mei-41, both in homozygous and heterozygous lines. In mei-41 line males' lives longer than females in contrast to other lines.     

A cell-cycle stage-related chromosomal X-ray hypersensitivity in larval neuroblasts of Drosophila mei-9 and mei-41 mutants suggesting defective DNA double-strand break repair

We have examined the chromosomal X-ray hypersensitivity in relation to the cell cycle in larval neuroblasts of the mutagen-sensitive and excision repair-defective mutant mei-9 and of the mutagen-sensitive and post-replication repair-defective mutant mei-41 of Drosophila melanogaster. When compared to wild-type cells, cells bearing the mei-9L1 allele produced unusually high levels in particular of chromatid deletions and to a lesser extent also of isochromatid deletions, but virtually no exchange aberrations. The chromosomal hypersensitivity is apparent at M1 when cells are irradiated in S or G2 but not when irradiated in G1. On the other hand, following irradiation cells bearing the mei-41D5 allele predominantly produce chromosome deletions. Also dicentric and chromatid exchange formation is enhanced with a moderate increase in chromatid deletions. The phases of major sensitivity are the S and G1. Mei-9 and mei-41 mutants have been classified to date as proficient in DNA double-strand break repair. The data presented in this paper revealed an S-independent clastogenic hypersensitivity of mei-9 and mei-41 cells. They are interpreted as indicative evidence for the presence of impaired DNA double-strand break repair. The cell-cycle-related difference in the ratio of chromatid- versus chromosome-type deletions in both mutants suggests repair defects at partially different phases of the cell cycle in mei-9 and mei-41 mutant cells.     

Studies on mutagen-sensitive strains of Drosophila melanogaster. IV. Modification of genetic damage induced by X-irradiation of spermatozoa and spermatids in N2 or O2 by mei-9a, mei-41D5 and mus(1)101D1

The influence of defects in DNA repair processes on X-ray-induced genetic damage in post-meiotic male germ cell stages of Drosophila melanogaster was studied using the 'maternal effects approach'. Basc males were irradiated in N2, air or O2 either as 48-h-old pupae (to sample spermatids) or as 3-4-day-old adults (to sample mature spermatozoa) and mated to females of 3 repair-deficient strains (mei-9a: excision-repair-deficient; mei-41D5: post-replication-repair-deficient; mus(1)101D1: post-replication-repair-deficient and impaired in DNA synthesis). Simultaneous controls involving mating of males to repair-proficient females (mei+) were run. The frequencies of sex-linked recessive lethals and of autosomal translocations were determined following standard genetic procedures. The responses elicited in the different crosses with repair-deficient females were compared with those in mei+ crosses. The main findings are the following: with mei-9 females, the frequencies of recessive lethals are higher after irradiation of spermatids in N2, but not after irradiation in air of O2 (relative to those in the mei+ crosses); this result is different from that obtained in earlier work with spermatozoa, in which cell stage, higher yields of recessive lethals were obtained after irradiation of males in either N2 or air; in the mei-9 crosses, there are no significant differences in response (relative to mei+) after irradiation of either spermatozoa or spermatids in O2; the translocation frequencies in the mei-9 crosses are similar to those in the mei+ crosses, irrespective of the treated germ cell stage or the irradiation atmosphere; irradiation of either spermatozoa or spermatids in N2, air or O2 does not result in any differential recovery of recessive lethals in the mei-41 relative to mei+ crosses; irradiation of spermatids in N2 and of spermatozoa in air leads to a higher recovery of translocations in the mei-41 crosses; and after irradiation of spermatids or spermatozoa in any of the gaseous atmospheres, the frequencies of recessive lethals and of translocations are lower in the mus-101 crosses. The differences in responses (between cell stages, in different gaseous atmospheres and with different repair-deficient females) are explained on the basis of both qualitative and quantitative differences in the composition of the initial lesions and the extent to which their repair may be affected by the defects present in the different repair-deficient females. Several discrepancies between expectations based on biochemical results and the genetic results are pointed out.(ABSTRACT TRUNCATED AT 400 WORDS)     

DNA Repair Dependence of Somatic Mutagenesis of Transposon-Caused WHITE Alleles in DROSOPHILA MELANOGASTER after Treatment with Alkylating Agents

DNA repair-defective alleles of the mei-9, mei-41, mus-104 and mus-101 loci of Drosophila melanogaster were introduced into stocks bearing the UZ and SZ marker sets. Males with the UZ marker set, z1 (zeste allele) and w+(TE) (genetically unstable white allele presumably caused by a transposable element), or the SZ marker set, z1 and w+R (semistable white allele caused by partial duplication of the w+ locus plus transposon insert), were exposed to EMS at the first instar. After emergence, adult males bearing red spots on lemon-yellow eyes were scored as flies with somatic reversions of w+(TE) or w+R. The relative mutabilities (relative values of reversion frequency at an equal EMS dose) of either w+(TE) or w+R in a repair-proficient strain and in mei-9, mei-41, mus-104 and mus-101 strains were 1: approximately 1.2:0.3:0.3:0.7, despite the fact that w+(TE) reverted two to three times as frequently as w+R under both the repair-proficient and repair-deficient genetic conditions. Similarly, after treatment with MMS, MNNG and ENNG, w+(TE) was somatically more mutable in the mei-9 strain and less mutable in the mei-41 and mus-104 strains than in the repair-proficient strain. From these results, we propose that mutagenic lesions produced in DNA by treatment with these chemicals are converted to mutant DNA sequences via the error-prone repair mechanisms dependent on the products of the genes mei-41+ (mei-41 and mus-104 being alleles of the same locus) and mus-101+, whereas they are eliminated by mei-9+-dependent excision repair. In contrast to the approximately linear responses of induced reversions of w+(TE) with ENNG in the repair-proficient, mei-9, and mei-41 strains, seemingly there were dosage insensitive ranges for induced reversion with MNNG in the repair-proficient and mei-41 strains, but not for reversion in the mei-9 strain; w+(TE) in the mus-104 strain was virtually nonmutable with MNNG and ENNG. These results suggest that O6-methylguanine (O6MeG) produced in DNA with MNNG, but not O6-ethylguanine produced with ENNG, is almost completely repaired in a low dose range by constitutive activity of DNA O6MeG transmethylase. From the distribution of clone sizes of spontaneous revertant spots and other data, we propose that both w+(TE) and w+R have a similar tendency to spontaneously revert more frequently at early rather than at later developmental stages probably reflecting a common property of their inserted transposons.     

Genetic study on the effects of the repair-deficient mutant females mei-9a, mei-41D5, mus101D1, mus104D1 and mus302D1 of Drosophila on spontaneous and X-ray-induced chromosome loss in the paternal genome

The repair-deficient mutants mei-9a, mei-41D5, mus101D1, mus104D1 and mus302D1 in Drosophila melanogaster were investigated regarding their effects on spontaneous and X-ray-induced chromosome loss in postmeiotic cells. Each mutant was incorporated singly into XC2, and the ring-X male provided with BSYy+. From matings of males carrying mus101D1, mus302D1 or mei-41D5, mutants identifying a caffeine-sensitive (CAS) postreplication-repair pathway, with corresponding mutant females, and non-mutant males to non-mutant females, overall frequencies of spontaneous partial loss and spontaneous complete loss were significantly increased in each mutant cross except for spontaneous complete loss with mus302 where an increase was noted only in brood 2. Similar findings were noted when males carrying the excision-repair mutant mei-9a were mated with mei-9a females. Males carrying the mutant mus104D1, identifying a caffeine-insensitive (CIS) postreplication-repair pathway, tested with mus104D1 females, produced results that were not significantly different from non-mutant controls. When males were given 3000 rad X-irradiation, frequencies of induced partial loss were significantly higher with mus101D1, mus302D1, mei-41D5 and mei91, and not significantly higher with mus101D1, mus302D1, mei41D5 and mei-9a, and not significantly different from controls with mus104D1. It was suggested that the functional CAS postreplication-repair pathway primarily promotes repair of breaks while an alternative pathway(s) not defined by mus104 promotes misrepair. Therefore, the significant increases in both spontaneous and induced partial loss with the excision-repair-deficient mutant mei-9a suggests the possibility that (a) the excision-repair-pathway may not function in misrepair and (b) the undefined misrepair pathway may be dominant pathway for postreplication repair in Drosophila since mei-9a females presumably have functional postreplication repair and misrepair capacity. The suggestion that the CAS postreplication-repair pathway and the excision-repair pathway function primarily in repair, and an undefined pathway in misrepair is in line with the finding that with mus104D1, no significant increase was found in spontaneous complete loss, but with mus101D1, mus302D1, mei-41D5 and mei-9a significant increases were observed. Results on induced complete loss, with the exception of those with mei-41D5, show a poor correlation with other classes of loss of each of the mutants. Possible explanations for this discrepancy are discussed.     

Tracing the tracks of genotoxicity by trivalent and hexavalent chromium in Drosophila melanogaster

Mutagen sensitive strains (mus) in Drosophila are known for their hypersensitivity to mutagens and environmental carcinogens. Accordingly, these mutants were grouped in pre- and post-replication repair pathways. However, studying mutants belonging to one particular repair pathway may not be adequate for examining chemical-induced genotoxicity when other repair pathways may neutralize its effect. To test whether both pre-and post-replication pathways are involved and effect of Cr(III)- and Cr(VI)-induced genotoxicity in absence or presence of others, we used double mutant approach in D. melanogaster. We observed DNA damage as evident by changes in Comet assay DNA migration in cells of larvae of Oregon R(+) and single mutants of pre- (mei-9, mus201 and mus210) and post- (mei-41, mus209 and mus309) replication repair pathways and also in double mutants of different combinations (pre-pre, pre-post and post-post replication repair) exposed to increasing concentrations of Cr(VI) (0.0, 5.0, 10.0 and 20.0 μg/ml) for 48 h. The damage was greater in pre-replication repair mutants after exposure to 5.0 μg/ml Cr(VI), while effects on Oregon R(+) and post replication repair mutants were insignificant. Post-replication repair mutants revealed significant DNA damage after exposure to 20.0 μg/ml Cr(VI). Further, double mutants generated in the above repair categories were examined for DNA damage following Cr(VI) exposure and a comparison of damage was studied between single and double mutants. Combinations of double mutants generated in the pre-pre replication repair pathways showed an indifferent interaction between the two mutants after Cr(VI) exposure while a synergistic interaction was evident in exposed post-post replication repair double mutants. Cr(III) (20.0 μg/ml) exposure to these strains did not induce any significant DNA damage in their cells. The study suggests that both pre- and post-replication pathways are affected in Drosophila by Cr(VI) leading to genotoxicity, which may have consequences for metal-induced carcinogenesis.     

Mutagen-sensitive mutants in Drosophila melanogaster: effects on premutational damage.

Drosophila melanogaster males from a Basc stock were mutagenized with either X-rays, ethyl methanesulfonate (EMS), or nitrogen mustard (HN2). Groups of identically treated males were crossed to different types of female. Sex-linked recessive lethals were scored as a genetic end point. The females used were homozygous for X-chromosomal mutations (mus(1)101D1, mus(1)104D1, mei-9 or mei-41D5) which lead to defective DNA repair and which increase the mutagen sensitivity of larvae. Females from a white stock with normal DNA repair capacities served as controls. The premutational lesions induced in mature sperm are only processed after insemination by the maternal enzyme systems present in the oocytes. Differences in the efficiency of the processing of lesions can lead to maternal effects on the frequency of mutations recovered from mutagenized sperm. It was found that, with the exception of mus(1)104D1, all mutants analysed significantly modify the mutation fixation of one or more types of premutational lesions. The most drastic effect is found with the mus(1)101D1 stock in which HN2-induced DNA cross-links do not lead to sex-linked recessive lethals. It is assumed that mus(1)101D1 is defective in an early step of DNA cross-link repair. Our first set of data clearly demonstrates that the study of maternal effects in Drosophila is an efficient tool to analyse the in vivo function of repair mutations on chemically induced mutagenesis.     

Effect of the yellow locus on sensitivity of drosophila sex cells to chemical mutagens

The effect of the yellow (y) locus on germ cell sensitivity to the alkylating agent ethyl methanesulfonate (EMS) has been studied in Drosophila. Since DNA repair is one of the most important factors that control cell sensitivity to mutagens, the approaches used in our experiments aimed at evaluating the relationship between germ-cell mutability and activity of DNA repair. Germ-cell mutability and repair activity were assessed using several parameters, the most important of which was the frequency of the recessive sex-linked lethal mutations (RSLLM). In one series of experiments, the adult males of various genotypes (Berlin wild; y; y ct v; y mei-9a) were treated by mutagenic agents and then crossed to Basc females. Comparative analysis of germ-cell mutability as dependent on genotype and the stage of spermatogenesis showed that the yellow mutation significantly enhanced the premeiotic cell sensitivity to EMS, presumably, due to the effect on DNA repair. In the second series of experiments, the effect of the maternal DNA repair was studied and, accordingly, mutagen-treated Basc males were crossed to females of various genotypes including y and y mei-9a ones. The crosses involving y females yielded F1 progeny with high spontaneous lethality, whereas in F2, the frequency of spontaneous mutations was twice higher. The germ cell response to EMS depended also on female genotype: the effect of yellow resulted in increased embryonic and postembryonic lethality, whereas the RSLLM frequency decreased insignificantly. The latter result may be explained by elimination of some mutations due to 50% mortality of the progeny. The results obtained using the above two approaches suggest that the yellow locus has a pleiotropic effect on the DNA repair systems in both males and females of Drosophila.     

The relationship between reaction kinetics and mutagenic action of monofunctional alkylating agents in higher eukaryotic systems. IV. The effects of the excision-defective mei-9L1 and mus(2)201D1 mutants on alkylation-induced genetic damage in Drosophila

Repair-defective mutants of Drosophila melanogaster which identify two major DNA excision repair loci have been examined for their effects on alkylation-induced mutagenesis using the sex-linked recessive lethal assay as a measure of genotoxic endpoint. The alkylating agents (AAs) chosen for comparative analysis were selected on the basis of their reaction kinetics with DNA and included MMS, EMS, MNU, DMN, ENU, DEN and ENNG. Repair-proficient males were treated with the AAs and mated with either excision-defective mei-9L1 or mus(2)201D1 females or appropriate excision-proficient control females. The results of the present work suggest that a qualitative and quantitative relationship exists between the nature and the extent of chemical modification of DNA and the induction of of genetic alterations. The presence of either excision-defective mutant can enhance the frequency of mutation (hypermutability) and this hypermutability can be correlated with the Swain-Scott constant S of specific AAs such that as the SN1 character of the DNA alkylation reaction increases, the difference in response between repair-deficient and repair-proficient females decreases. The order of hypermutability of AAs with mei-9L1 relative to mei-9+ is MMS greater than MNU greater than DMN = EMS greater than iPMS = ENU = DEN = ENNG. When the percentage of lethal mutations induced in mei-9L1 females are plotted against those determined for control females, straight lines of different slopes are obtained. These mei-9L1/mei-9+ indices are: MMS = 7.6, MNU = 5.4, DMN = 2.4, EMS = 2.4 and iPMS = ENU = DEN = ENNG = 1. An identical order of hypermutability with similar indices is obtained for the mus(2)201 mutants: MMS(7.3) greater than MNU (5.4) greater than EMS(2.0) greater than ENU(1.1). Thus, absence of excision repair function has a significant effect on mutation production by AAs efficient in alkylating N-atoms in DNA but no measurable influence on mutation production by AAs most efficient in alkylating O-atoms in DNA. The possible nature of these DNA adducts has been discussed in relation to repair of alkylated DNA. In another series of experiments, the effect on alkylation mutagenesis of mei-9L1 was studied in males, by comparing mutation induction in mei-9L1 males vs. activity in Berlin K (control). Although these experiments suggested the existence of DNA repair in postmeiotic cells during spermatogenesis, no quantitative comparisons could be made.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genetic damage induced by X-rays or neutrons in spermatozoa of Drosophila melanogaster; differential processing in the oocytes of females carrying the DNA-repair-deficient mutants mei-9a and mus-101d1

Radiation-induced premutational lesions on the chromosomes of irradiated mature spertozoa of Drosphila are processed when the sperm nucleus the egg cytoplasm at fertilization. This processing depends on enzymatic repair systems, which are built up in ocytes under the control of the maternal genotype. The present study is concerned with 2 repair-deficient mutants, mei-9^a and mus-101^D1. Irradiated Basc males were crossed to homozygous mei-9^a or mus-101^D1 females, or to repair-proficient control females. The frequencies of recovered sex-link recessive lethal mutations and of II–III translocations were used to assess the effects of impaired maternal repair. Neutrons, as a densely ionizing radiation, and X-rays as a sparsely ionizing one, were used to induce the premutational lesions.The question being asked was whether different radiation qualities cause specific types of lesion that are processed differentially under conditions of impaired maternal repair. The results indicate that this may be so. In comparison with the control, with repair-proficient females, all major effects caused by impaired maternal repair led to frequency reductions in the recovery of lethals and translocations. These reductions in yield were pronounced in all neutron experiments, whereby mus-101^D1 had a stronger effect than mei-9^a. Two possible explanations are considered. The first is based on the idea that specific lesions are processed in a specific way, resulting in a specific mutational end-product, which may not be recovered when repair is impaired. The second is based on the notion that energy deposition in cells exposed to neutrons is not uniform, which leads to clustered damage. Impaired repair may select againts multiply damaged cells much more powerfully than normal repair. Consequently, the surviving fraction of cells is likely to have received less than the average dose. With X-rays, no or only spurious effects of the repair-defective mutants were detected, except in the following case: recovery of translocations (but not of lethals) was strongly reduced when irradiated males were crossed to mus-101^D1 females. It is assumed that mus-101^D1 is defective in repair of DNA double-strand damage, and that the formation of translocations may depend particularly on this repair function.     

Studies on mutagen-sensitive strains of Drosophila melanogaster. VI. The effect of DNA-repair deficiencies in spermatids, spermatocytes and spermatogonia irradiated in N2 or O2

This study was aimed at ascertaining the extent to which paternal repair processes possibly deficient in mei-9a, mei-41D5 and mus-101D1 genotypes would affect the recovery of radiation-induced recessive lethals in early spermatids, spermatocytes and spermatogonia. These germ cell stages were sampled in two 2-day broods from freshly hatched males, that were irradiated as 24-h old pupae in O2, or N2 followed by N2 or O2 post-treatment. Spontaneous mutation frequencies were higher in mei-9 and mei-41 males, and thus appropriate corrections were applied to the radiation data. Only with mei-9 males a clear and consistent increase of the radiation-induced mutation frequency was observed. The effect is somewhat more pronounced in brood B, presumably representing spermatogonia, than in brood A and is observed after radiation in either O2 or N2. The paternal repair process thus differs from the maternal one in that it also responds to radiation damage induced in O2. The finding that, following irradiation under anoxia, post-treatment with O2 (versus that with N2), also lowers the mutation frequency in mei-9 males, indicates that the repair defect in mei-9 does not interfere with oxygen-dependent post-radiation repair. Thus there are two different paternal repair processes in these early stages of spermatogenesis: that is, one controlled by mei-9 and one depending on oxygen. Mei-41 and mus-101 do not appear to interfere with the paternal repair process. The frequency of translocations recovered from these stages was likewise not affected by mus-101.     

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, we generated Drosophila lines 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. We also found that Claspin was phosphorylated in response to HU and IR treatment and a hyperphosphorylated form of Claspin was generated only after HU treatment in mei-41/ATR-dependent and tefu/ATM-independent way. In summary, our 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.     

The meiotic-9 (mei-9) mutants of Drosophila melanogaster are deficient in repair replication of DNA

Summary Repair replication of DNA has been studied in first instar larvae and cultured cells of meiotic-9 (mei-9) mutants in Drosophila melanogaster. Results obtained with both experimental systems show that the mei-9 mutants are defective in this form of repair after UV and X-ray treatment. This defect is correlated with the observation that male larvae carrying alleles of this locus are hypersensitive to killing by UV and X-rays. These findings strengthen the suggestion derived from genetic data that the normal mei-9 ⁺ function is involved in the exchange process of meiotic recombination rather than in a precondition to exchange (Carpenter and Sandler, 1974).Abbreviations FdUrd 5-fluorodeoxyuridine - BrdUrd 5-bromodeoxyuridine - ³H-dThd thymidine methyl-³H - SSC 0.15 M sodium chloride-0.015 M sodium citrate - UV ultraviolet radiation-predominantly 254 nm     

The interaction of the rad201 gene with mei-9 and mei-41 genes in germline cells of Drosophila female

The effect of Drosophila mutation rad201G1 together with mutations mei-41D5 and mei-9a on the sensitivity of oocytes to induction of dominant lethals (DLs) was studied. To this end, the frequencies of spontaneous and gamma-radiation-induced DLs in consecutive egg batches of females carrying double or single mutations were estimated. Since the effects of the mutations examined are expressed only at the previtellogenetic stages of oogenesis, only newly hatched (0-5-hour-old) females, whose oocytes did not develop farther than stage 7, were irradiated. The results obtained indicated that in intact and irradiated oocytes of double mutants mei-9a rad201G1 and mei-41D5 rad201G1, mutation rad201G1 epistatically suppresses the mutations of the both mei genes.     

The mei-9a Mutant of DROSOPHILA MELANOGASTER Increases Mutagen Sensitivity and Decreases Excision Repair

The mei-9^a mutant of Drosophila melanogaster , which reduces meiotic recombination in females (Baker and Carpenter 1972), is deficient in the excision of UV-induced pyrimidine dimers in both sexes. Assays were performed in primary cultures and established cell lines derived from embryos. An endonuclease preparation from M. luteus , which is specific for pyrimidine dimers, was employed to monitor UV-induced dimers in cellular DNA. The rate of disappearance of endonuclease-sensitive sites from DNA of control cells is 10–20 times faster than that from mei-9^a cells. The mutant mei-218, which is also deficient in meiotic recombination, removes nuclease-sensitive sites at control rates. The mei-9^a cells exhibit control levels of photorepair, postreplication repair and repair of single strand breaks. In mei-9 cells DNA synthesis and possibly postreplication repair are weakly sensitive to caffeine. Larvae which are hemizygous for either of the two mutants that define the mei-9 locus are hypersensitive to killing by the mutagens methyl methanesulfonate, nitrogen mustard and 2-acetylaminofluorene. Larvae hemizygous for the mei-218 mutant are insensitive to each of these reagents. These data demonstrate that the mei-9 locus is active in DNA repair of somatic cells. Thus functions involved in meiotic recombination are also active in DNA repair in this higher eukaryote. The results are consistent with the earlier suggestions (Baker and Carpenter 1972; Carpenter and Sandler 1974) that the mei-9 locus functions in the exchange events of meiosis. The mei-218 mutation behaves differently in genetic tests and our data suggest its function may be restricted to meiosis. These studies demonstrate that currently recognized modes of DNA repair can be efficiently detected in primary cell cultures derived from Drosophila embryos.     

The Drosophila ATM ortholog, dATM, mediates the response to ionizing radiation and to spontaneous DNA damage during development

Cells of metazoan organisms respond to DNA damage by arresting their cell cycle to repair DNA, or they undergo apoptosis. Two protein kinases, ataxia-telangiectasia mutated (ATM) and ATM and Rad-3 related (ATR), are sensors for DNA damage. In humans, ATM is mutated in patients with ataxia-telangiectasia (A-T), resulting in hypersensitivity to ionizing radiation (IR) and increased cancer susceptibility. Cells from A-T patients exhibit chromosome aberrations and excessive spontaneous apoptosis. We used Drosophila as a model system to study ATM function. Previous studies suggest that mei-41 corresponds to ATM in Drosophila; however, it appears that mei-41 is probably the ATR ortholog. Unlike mei-41 mutants, flies deficient for the true ATM ortholog, dATM, die as pupae or eclose with eye and wing abnormalities. Developing larval discs exhibit substantially increased spontaneous chromosomal telomere fusions and p53-dependent apoptosis. These developmental phenotypes are unique to dATM, and both dATM and mei-41 have temporally distinct roles in G2 arrest after IR. Thus, ATM and ATR orthologs are required for different functions in Drosophila; the developmental defects resulting from absence of dATM suggest an important role in mediating a protective checkpoint against DNA damage arising during normal cell proliferation and differentiation.     

Studies on mutagen-sensitive strains of Drosophila melanogaster. II. Detection of qualitative differences between genetic damage induced by X-irradiation of mature spermatozoa in oxygenated and anoxic atmospheres through the use of the repair-deficient mutant mei-9a

Muller-5 males were irradiated with X-rays in nitrogen, in air or in oxygen (followed by nitrogen or oxygen post-treatments in the nitrogen and oxygen series) and were mated to females of a repair-proficient strain (mei+) or to those of a strain known to be deficient in excision repair of UV damage (in somatic cells). The latter strain, designated as mei-9a, is also known to be sensitive, in the larval stages, to the killing effects of UV, X-rays and to a number of chemical mutagens. The frequencies of sex-linked recessive lethals and autosomal translocations induced in the spermatozoa of males were determined and compared. The frequencies of sex-linked recessive lethals in the mei-9 control groups were consistently higher than in the mei+ groups. Irradiation in air or in nitrogen led to significantly higher yields of recessive lethals when the irradiated males were mated to mei-9 females, whereas, after irradiation in oxygen, the yields were similar with both kinds of female. No significant differences in the frequencies of reciprocal translocations were observed between the mei+ and mei-9 groups after irradiation of the males in nitrogen, in air or in oxygen. Likewise, no differential effects of the contrasting post-treatments (nitrogen versus oxygen), either for recessive lethals or for translocations, could be discerned. These results are considered to support the notion that the kinds of genetic damage induced in mature spermatozoa in air or in nitrogen are qualitatively similar (at least with respect to the component(s) that lead to the production of recessive lethal mutations), but clearly different when induced in an oxygen atmosphere. The enhanced yields of recessive lethals with mei-9 females (after irradiation of the males either in air or in nitrogen) has been interpreted on the assumption that the mei-9 mutant is also deficient for the repair of X-ray-induced, recessive lethal-generating premutational lesions. Possible reasons for the lack of differences between the mei+ and mei-9 groups with respect to translocation yields and for the absence of measurable differences in response between the contrasting post-treatments (after irradiation of the males in nitrogen) are discussed.     

Induced chromosome loss following treatment of postmeiotic cells of the Drosophila melanogaster male with MMS and DMN and matings with repair-proficient females and the repair-deficient females mei-9a and st mus302

Drosophila melanogaster ring-X males carrying a double marked Y chromosome, BsYy+, were treated with MMS or DMN and mated with repair-proficient females or the repair-deficient females mei-9a and st. mus302. Frequencies of induced complete loss (principally the ring-X) and partial losses of the Y chromosome (loss of Bs or Y+) decreased in the sequence st must302 greater than mei-9a greater than repair-proficient females agreeing with the sequence obtained previously with procarbazine and DEN. With MMS and DMN, some 30-40% or more or partial Y chromosome losses are mosaics from mei-9a and only 0.4% from st mus302 females and a delay in mei-9a females. Similar findings with procarbazine and DEN are indicated. That the higher sensitivity of st mus302 relative to mei-9a results from impairments in both postreplication and excision repair in the former remains to be determined.     

O-alkylation in DNA does not correlate with the formation of chromosome breakage events in D. melanogaster

Postmeiotic cell stages of repair-proficient ring-X (RX) males were treated with methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), diethylnitrosamine (DEN) or ethylnitrosourea (ENU) and then mated to either repair-defective (mei-9L1) or to repair-competent females (mei-9+). Absence of the mei-9+ function resulted in a hypermutability effect to all alkylating agents (AAs) when they were assayed for their ability to induce chromosomal aberrations (chromosome loss; CL), irrespective of marked differences in distribution of DNA adducts brought about by these AAs. This picture is different from that described previously for the induction of point mutations (Vogel et al., 1985a). There, evidence was presented indicating that reduction in DNA excision repair does not affect point mutation induction (recessive lethals) by those AAs most efficient in ring-oxygen alkylation such as ENU, DEN, N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), and isopropyl methanesulfonate (iPMS): the order of hypermutability of AAs with mei-9L relative to mei-9+ was MMS greater than MNU greater than DMN = EMS greater than iPMS = ENU = DEN = ENNG. When the percentage of lethal mutations induced in mei-9L1 females were plotted against those determined for mei-9+ females, straight lines of following slopes were obtained: MMS = 7.6, MNU = 5.4, DMN = 2.4, EMS = 2.4, and iPMS = ENU = DEN = ENNG = 1. Those findings, together with the recent observation that AAs do not split into two groups when assayed for their ability to cause CL, point to the involvement of different DNA alkylation products in ENU- and DEN-induced chromosome loss vs. that of point mutations. It is concluded that with ENU and DEN chromosomal loss results from N-alkylation products whereas point mutations (SLRL) are the consequence of interactions with oxygen-sites in DNA. Thus, as a consequence of a very dominating role of O-ethylguanine (and possibly O4-alkylation of thymine), N-alkylation in DNA does not contribute measurably to mutation induction in the case of ENU-type mutagens while O-alkylation, very clearly, does not show a positive correlation with the formation of chromosome breakage events in Drosophila. Conversely, it appeared that with MMS-type mutagens (MMS; dimethyl sulfate, DMS; trimethyl phosphate, TMP), alkylation products such as 7-methylguanine and 3-methyladenine, if unrepaired or misrepaired, are potentially mutagenic lesions causing both mutations and chromosomal aberrations.     

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

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: DNA repair is essential for surviving radiation as expected; surprisingly, cell cycle regulation and p53-dependent cell death are not. We propose that processes resembling regeneration of discs act to maintain tissues and ultimately determine survival after irradiation, thus distinguishing requirements between muticellular and unicellular eukaryotes.