Abnormal recovery of DNA replication in ultraviolet-irradiated cell cultures of Drosophila melanogaster which are defective in DNA repair

Summary Cell cultures prepared from embryos of a control stock of Drosophila melanogaster respond to ultraviolet light with a decline and subsequent recovery both of thymidine incorporation and in the ability to synthesize nascent DNA in long segments. Recovery of one or both capacities is absent or diminished in irradiated cells from ten nonallelic mutants that are defective in DNA repair and from four of five nonallelic mutagen-sensitive mutants that exhibit normal repair capabilities. Recovery of thymidine incorporation is not observed in nine of ten DNA repair-defective mutants. On the other hand, partial or complete recovery of incorporation is observed in all but one repair-proficient mutagen-sensitive mutant.Irradiated cells from two mutants that display no excision capacity exhibit a gradual arrest of thymidine incorporation within 20 h after the initial decline. This arrest of incorporation is not observed in mutants exhibiting only partial defects in excision repair.Recovery of the ability to synthesize nascent DNA in long segments is normal in cells from the two mutants that display no excision capacity, indicating that recovery does not depend upon the excision of pyrimidine dimers from cellular DNA. Recovery of that ability is not observed, however, in cells from one partially excision-defective mutant, two of three postreplication repair-defective mutants, two of four mutants defective in both excision and postreplication repair, and one of five repair-proficient mutagen-sensitive mutants. These results indicate that recovery of normal DNA replication in irradiated Drosophila cells depends upon the activity of several functions.Abbreviation used UV ultraviolet light — principal wavelength 254 nm     

Hypersensitivity of Drosophila mei-41 mutants to hydroxyurea is associated with reduced mitotic chromosome stability

6 mutant alleles of the mei-41 locus in Drosophila melanogaster are shown to cause hypersensitivity to hydroxyurea in larvae. The strength of that sensitivity is directly correlated with the influence of the mutant alleles on meiosis in that: alleles exhibiting a strong meiotic effect (mei-41D2, mei-41D5, mei-41D7) are highly sensitive; alleles with negligible meiotic effects (mei-41(104)D1, mei-41(104)D2) are moderately sensitive and an allele which expresses meiotic effects only under restricted conditions (mei-41D9) has an intermediate sensitivity. This sensitivity is not a general feature of strong postreplication repair-deficient mutants, because mutants with that phenotype from other loci do not exhibit sensitivity (mus(2)205A1, mus(3)302D1, mus(3)310D1). The observed lethality is not due to hypersensitivity of DNA synthesis in mei-41 larvae to hydroxyurea as assayed by tritiated thymidine incorporation. Lethality is, however, potentially attributable to an abnormal enhancement of chromosomal aberrations by hydroxyurea in mutant mei-41 larvae. Both in vivo and in vitro exposure of neuroblast cells to hydroxyurea results in an increase in 3 types of aberrations which is several fold higher in mei-41 tissue. Since hydroxyurea disrupts DNA synthesis, these results further implicate the mei-41 locus in DNA metabolism and provide an additional tool for an elucidation of its function. The possible existence of additional genes of this nature is suggested by a more modest sensitivity to hydroxyurea which has been detected in two stocks carrying mutagen-sensitive alleles of alternate genes.     

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.     

P transposition in Drosophila provides a new tool for analyzing postreplication repair and double-strand break repair.

A genetic screen has been developed in Drosophila for identifying host-repair genes responsible for processing DNA lesions formed during mobilization of P transposable elements. Application of that approach to repair deficient mutants has revealed that the mei-41 and mus302 genes are necessary for recovery of P-bearing chromosomes undergoing transposition. Both of these genes are required for normal postreplication repair. Mutants deficient in excision repair, on the other hand, have no detected effect on the repair of transposition-induced lesions. These observations suggest that P element-induced lesions are repaired by a postreplication pathway of DNA repair. The data further support recent studies implicating double-strand DNA breaks as intermediates in P transposition, because the mei-41 gene has been genetically and cytologically associated with the repair of interrupted chromosomes. Analysis of this system has also revealed a striking stimulation of site-specific gene conversion and recombination by P transposition. This result strongly suggests that postreplication repair in this model eukaryote operates through a conversion/recombination mechanism. Our results also support a recently developed model for a conversion-like mechanism of P transposition (Engels et al., 1990). Involvement of the mei-41 and mus302 genes in the repair of P element-induced double-strand breaks and postreplication repair points to a commonality in the mechanisms of these processes.     

Postreplication repair defects in mutants of Drosophila melanogaster

Summary Mutants of Drosophila melanogaster which are defective in DNA synthesis have been identified among mutagen-sensitive stocks through analysis of both organ and cell cultures. A new procedure employing larval brain ganglia allows poorly fertile or sterile mutants to be analyzed for the first time. Parallel studies were performed in both tissues to establish the sensitivity of the new assay relative to that of the proven cell-culture assay. Damage was induced in the DNA of cultured cells with UV irradiation and in that of ganglial cells with the carcinogen N-acetoxy-2-acetylaminofluorene. Cultures were then pulse-labeled with ³H-thymidine, incubated in the absence of thymidine, and the newly synthesized DNA was analyzed by alkaline sucrose gradient centrifugation. The molecular weight of labeled DNA from mutant cells was compared with that from control cells to assess the effect of the mutant on DNA synthesis. Among 16 mutant stocks that were scanned in either or both tissues, seven show reductions in DNA synthesis using an undamaged template. Mutants at five different genetic loci [mus(2)205, mus(3)304, mus(3)308, mus(3)310 and mus(3)311] possess a reduced capacity to synthesize DNA on a UV-damaged template in primary cell cultures. Four of these five defects can also be detected in carcinogen-treated organ cultures. Two additional defects in postreplication repair were observed with the brainganglia assay in strains that cannot be assayed in cell culture [mus(1)108, mus(2)206].Abbreviations MMS methyl methanesulfonate - HN2 nitrogen mustard - AAF 2-acetylaminofluorene - AAAF N-acetoxy-2-acetylaminofluorene - DMSO dimethyl sulfoxide     

Mutations at the mei-41, mus(1)101, mus(1)103, mus(2)205 and mus(3)310 loci of Drosophila exhibit differential UDS responses with different DNA-damaging agents

5 mutagen-sensitive mutants of Drosophila melanogaster, reported to perform normal or only slightly reduced excision repair of UV damage, were examined by an unscheduled DNA synthesis (UDS) assay. This assay measures the ability of cultured primary cells, derived from each mutant, to perform the resynthesis step in the excision repair pathway, following damage to cellular DNA by direct-acting alkylating agents, UV or X-irradiation. 2 mutants, classified as completely or partially proficient for both excision and postreplication repair of UV damage, mus(1)103 and mus(2)205, were found to give positive UDS responses only for UV damage. These mutants exhibit no measurable UDS activity following DNA damage by several different alkylating agents and X-rays. 3 mutants, classified as having no defect in excision repair, but measurable defects in postreplication repair of UV damage, mei-41, mus(1)101, and mus(3)310 exhibit 3 different response patterns when tested with the battery of agents in the UDS assay. The mutant mei-41 exhibits a highly positive UDS response following damage by all agents, consistent with its prior classification as excision-repair-proficient, but postreplication-repair-deficient for UV damage. The mutant mus(1)101, however, exhibits a strong positive UDS response following only UV damage and appears to be blocked in the excision repair of damage produced by both alkylating agents and X-irradiation. Finally, mus(3)310 exhibits no UDS response to alkylation, X-ray or UV damage. This is not consistent with its previous classification. Results obtained with the quantitative in vitro UDS assay are entirely consistent with the results from two separate in vivo measures of excision repair deficiency following DNA damage, larval hypersensitivity to killing and hypermutability in the sex-linked recessive lethal test.     

Identification of a Second Locus in DROSOPHILA MELANOGASTER Required for Excision Repair

The mus(2)201 locus in Drosophila is defined by two mutant alleles that render homozygous larvae hypersensitive to mutagens. Both alleles confer strong in vivo somatic sensitivity to treatment by methyl methanesulfonate, nitrogen mustard and ultraviolet radiation but only weak hypersensitivity to X-irradiation. Unlike the excision-defective mei-9 mutants identified in previous studies, the mus(2)201 mutants do not affect female fertility and do not appear to influence recombination proficiency or chromosome segregation in female meiocytes.—Three independent biochemical assays reveal that cell cultures derived from embryos homozygous for the mus(2)^D1 allele are devoid of detectable excision repair. 1. Such cells quantitatively retain pyrimidine dimers in their DNA for 24 hr following UV exposure. 2. No measurable unscheduled DNA synthesis is induced in mutant cultures by UV treatment. 3. Single-strand DNA breaks, which are associated with normal excision repair after treatment with either UV or N-acetoxy-N-acetyl-2-aminofluorene,* are much reduced in these cultures. Mutant cells possess a normal capacity for postreplication repair and the repair of single-strand breaks induced by X-rays.     

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     

Molecular cloning of mei-41, a gene that influences both somatic and germline chromosome metabolism of Drosophila melanogaster

The mei-41 gene of Drosophila melanogaster plays an essential role in meiosis, in the maintenance of somatic chromosome stability, in postreplication repair and in DNA double-strand break repair. This gene has been cytogenetically localized to polytene chromosome bands 14C4-6 using available chromosomal aberrations. About 60 kb of DNA sequence has been isolated following a bidirectional chromosomal walk that extends over the cytogenetic interval 14C1-6. The breakpoints of chromosomal aberrations identified within that walk establish that the entire mei-41 gene has been cloned. Two independently derived mei-41 mutants have been shown to carry P insertions within a single 2.2 kb fragment of the walk. Since revertants of those mutants have lost the P element sequences, an essential region of the mei-41 gene is present in that fragment. A 10.5 kb genomic fragment that spans the P insertion sites has been found to restore methyl methanesulfonate resistance and female fertility of the mei-41 ^D3 mutants. The results demonstrate that all the sequences required for the proper expression of the mei-41 gene are present on this genomic fragment. This study provides the foundation for molecular analysis of a function that is essential for chromosome stability in both the germline and somatic cells.This Paper is dedicated to the memory of Professor James B. Boyd     

High frequency of spontaneous Minute mutations detected in the F1 progeny of interstrain matings between a recombination-defective mei-9L1 and the y w m f/sc8(y+) Y BS; dp strain of Drosophila melanogaster

The yield of spontaneous Minute mutations was recorded in the F1 progeny of interstrain (reciprocal) and intrastrain matings between a recombination- and excision repair-defective mei-9L1 (mei-9) strain and the y w m f/sc8(y+) Y BS; dp (ywmf-2) strain of Drosophila melanogaster. As a comparison, interstrain matings between a postreplication repair-defective st mus(3)302D1 (mus(3)) strain and the ywmf-2 strain were also studied for Minute mutations. The results show that: (1) a strikingly high frequency of Minute mutations is observed in the progeny of mei-9 female X ywmf-2 male crosses, but not in that of ywmf-2 females X mei-9 males; (2) no such difference exists in the progeny of intrastrain matings; and (3) there exists no marked inequality of Minute frequencies in the progeny of reciprocal crosses of mus(3) and ywmf-2 strains.     

Drosophila mutations at the mei-9 and mus(2)201 loci which block excision of thymine dimers also block induction of unscheduled DNA synthesis by methyl methanesulfonate, ethyl methanesulfonate, N-methyl-N-nitrosourea, UV light and X-rays

The mei-9 and mus(2)201 mutants of Drosophila melanogaster were identified as mutagen-sensitive mutants on the basis of larval hypersensitivity to methyl methanesulfonate and characterized as excision repair-deficient on the basis of a greatly reduced capacity to excise thymine dimers from cellular DNA. The high degree of larval cytotoxicity observed with a variety of other chemical and physical agents indicated that these mutants may be unable to excise other important classes of DNA adducts. We have measured the ability of the single mutants and the double mutant combination mei-9;mus(2)201 to perform the resynthesis step in excision repair by means of an autoradiographic analysis of unscheduled DNA synthesis (UDS) induced in a mixed population of primary cells in culture. The 3 strains exhibit no detectable UDS activity in response to applied doses of 1.5-6.0 mM methyl methanesulfonate, 1.0-4.5 mM N-methyl-N-nitrosourea or 10-40 J/m2 254-nm UV light, dose ranges in which control cells exhibit a strong dose-dependent UDS response. The mei-9 and mei-9;mus(2)201 mutants also have no detectable UDS response to X-ray doses of 300-1800 rad, whereas the mus(2)201 mutant exhibits a reduced, but dose-dependent, response over this range. These data correlate well with the degree of larval hypersensitivity of the strains and suggest that mutations at both loci block the excision repair of a wide variety of DNA damage prior to the resynthesis step.     

Postreplication repair in Neurospora crassa

Summary Changes in the molecular weight of nascent DNA made after ultraviolet (UV) irradiation have been studied in the excision-defective Neurospora mutant uvs-2 using isotopic pulse labeling, alkaline gradient centrifugation and alkaline filter elution. Both the size of nascent DNA and the rate of incorporation of label into DNA was reduced by UV light in a dose dependent manner. However, this DNA repair mutant did recover the ability to synthesize control-like high molecular weight DNA 3 hours after UV treatment, although the rate of DNA synthesis remained depressed after the temporary block to elongation (or ligation) had been overcome. Photoreactivation partially eliminated the depression of DNA synthesis rate and UV light killing of cells, providing strong evidence that the effects on DNA synthesis and killing were caused by pyrimidine cyclobutane dimers. The caffeine inhibition repair studies performed were difficult to quantitate but did suggest either partial inhibition of a single repair pathway or alternate postreplication DNA repair pathways in Neurospora. No enhancement in killing was detected after UV irradiation when cells were grown on caffeine containing plates.     

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.     

Molecular cloning of mei-41, a gene that influences both somatic and germline chromosome metabolism of Drosophila melanogaster

The mei-41 gene of Drosophila melanogaster plays an essential role in meiosis, in the maintenance of somatic chromosome stability, in postreplication repair and in DNA double-strand break repair. This gene has been cytogenetically localized to polytene chromosome bands 14C4-6 using available chromosomal aberrations. About 60 kb of DNA sequence has been isolated following a bidirectional chromosomal walk that extends over the cytogenetic interval 14C1-6. The breakpoints of chromosomal aberrations identified within that walk establish that the entire mei-41 gene has been cloned. Two independently derived mei-41 mutants have been shown to carry P insertions within a single 2.2 kb fragment of the walk. Since revertants of those mutants have lost the P element sequences, an essential region of the mei-41 gene is present in that fragment. A 10.5 kb genomic fragment that spans the P insertion sites has been found to restore methyl methanesulfonate resistance and female fertility of the mei-41 ^D3 mutants. The results demonstrate that all the sequences required for the proper expression of the mei-41 gene are present on this genomic fragment. This study provides the foundation for molecular analysis of a function that is essential for chromosome stability in both the germline and somatic cells.     

Genetic characterization of the mei-41 locus in Drosophila melanogaster

Summary The mutagen-sensitive mutant mus(1)104 ^D1 of Drosophila melanogaster maps to a position on the X chromosome very close to the meiotic mutant mei-41 ^D5 . Both mutants have been characterized as mutagen-sensitive and defective in post-replication repair. In the present report we show by complementation studies that mus(1)104 and mus(1)103 are allelic with mei-41. In addition, two reported alleles of mus(1)104 lie between the mei-41 alleles A10 and D5. The size of the mei-41 locus is estimated to be about 0.1 centimorgans (cM). Because several alleles of mei-41 have been shown to reduce recombination and increase meiotic chromosome loss and nondisjunction, mus(1)104 ^D1 females were examined for defects in meiosis. Although there was no evidence for reduced recombination on the second chromosome in homozygous mus(1)104 ^D1 females, heterozygous mus(1)104 ^D1 /mei-41 ^>D5 and mus(1)104 ^D1 /deficiency females showed reduced levels of recombination. However, there was no evidence of an increase in nondijunction in these females.We dedicate this article to the memory of Larry Sandler, who passed away suddenly on February 7, 1987     

Genetic mechanisms of formation of radiation-induced instability of the genome and its transgenerational effects in the descendants of chronically irradiated individuals of Drosophila melanogaster

The article is devoted to the study of the role of intracellular mechanisms in the formation of radiation-induced genetic instability and its transgenerational effect in cells of different tissues of the descendants of Drosophila melanogaster mutant strains whose parents were exposed to chronic radiation (0.42 and 3.5 mGy/h). The level of DNA damage (alkali-labile sites (ALS), single-strand (SSB) and double-strand (DSB) breaks) in cells of somatic (nerve ganglia, imaginal discs) and generative (testis) tissues from directly irradiated animals and their unirradiated offspring was evaluated. Confident transgenerational instability (on the level of ALSs and SSBs), observed only in somatic tissues and only at the higher dose rate, is characteristic for mus209 mutant strains defective in excision repair and, less often, for mus205 and mus210 mutant strains. The greatest manifestation of radiation-induced genetic instability was found in evaluating the DSBs. Dysfunction of the genes mus205, mus304, mei-9 and mei-41, which are responsible for postreplicative repair, excision repair, recombination and control of the cell cycle, affects transgenerational changes in the somatic tissues of the offspring of parents irradiated in both low and high dose rates. In germ cells, the key role in maintaining genetic stability under chronic irradiation is played by the non-recombination postreplication repair mus101 gene. We revealed the tissue specificity of the radiation-induced effects, transgenerational transmission and accumulation of DNA damage to descendants of chronically irradiated animals.     

Characterization of postreplication repair in Saccharomyces cerevisiae and effects of rad6, rad18, rev3 and rad52 mutations

Summary Postreplication repair of nuclear DNA was examined in an excision defective haploid strain of yeast lacking mitochondrial DNA (ral ⁰). The size of the DNA synthesized in cells exposed to various fluences of ultraviolet light (UV) corresponds approximately to the average interdimer distance in the parental DNA. Upon further incubation of cells following exposure to 2.5 J/m², the DNA increases in size; by 4 h, it corresponds to DNA from uniformly labeled cells. The alkaline sucrose sedimentation pattern of DNA pulse labeled at various times after UV irradiation, for up to 4 h, does not change substantially, indicating that dimers continue to block DNA replication. A significant amount of postreplication repair requires de novo protein synthesis, as determined by its inhibition by cycloheximide. The rad6 mutant does not carry out postreplication repair, the rad18 and rad52 mutants show great inhibition while the rev3 mutation does not affect postreplication repair. Both recombinational and nonrecombinational repair mechanisms may function in postreplication repair and most of postreplication repair is error free.     

Characterization of established,cell lines derived from mutagen-sensitiveDrosophila strains

Summary Six established cell lines have been generated from embryos ofDrosophila melanogaster homozygous for different X-linked mutations. Four of these mutants, confer hypersensitivity to chemical mutagens in larvae. The cell lines derived from the two mutageninsensitive stocks, serve as controls in the analyses of DNA metabolism. One cell line (UCD-Dm-mei-9-2) is uniquely identified by a strong hypersensitivity to ultraviolet radiation. Another (UCD-Dm-mus104-1) expresses an enzyme variant not found in the other lines. The population doubling time for these cultures varies between 24 and 47 h. Labeling indices of 24.4 to 37.5% were found. The duration of the S phase in one of the control cell lines is estimated to be about 9 h. Karyotype stability was monitored for five lines over a period of about 1 y. In general these cultures each, became hypotetraploid with a preferential loss of the Y and fourth chromosomes. DNA synthesis in two of the lines fails to exhibit the pattern of sensitivity to mutagens or caffeine that is observed in the corresponding primary cultures. In primary cultures three classes of cells can be identified by autoradiography. About 50% of the cells label at a moderate rate, 20% do not label within the first 1.5 d of culture, and the remaining cells exhibit a burst of labeling shortly after the cultures are initiated. This research was supported by NIH Grants GM16298 and GM22221 and by DOE Contract AT(04-3)-34 PA 210.     

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.     

Characterization of Postreplication Repair in Mutagen-Sensitive Strains of DROSOPHILA MELANOGASTER

Mutants of Drosophila melanogaster, with suspected repair deficiencies, were analyzed for their capacity to repair damage induced by X-rays and UV radiation. Analysis was performed on cell cultures derived from embryos of homozygous mutant stocks. Postreplication repair following UV radiation has been analyzed in mutant stocks derived from a total of ten complementation groups. Cultures were irradiated, pulse-labeled, and incubated in the dark prior to analysis by alkaline sucrose gradient centrifugation. Kinetics of the molecular weight increase in newly synthesized DNA were assayed after cells had been incubated in the presence or absence of caffeine. Two separate pathways of postreplication repair have been tentatively identified by mutants derived from four complementation groups. The proposed caffeine sensitive pathway (CAS) is defined by mutants which also disrupt meiosis. The second pathway (CIS) is caffeine insensitive and is not yet associated with meiotic functions. All mutants deficient in postreplication repair are also sensitive to nitrogen mustard. The mutants investigated display a normal capacity to repair single-strand breaks induced in DNA by X-rays, although two may possess a reduced capacity to repair damage caused by localized incorporation of high specific activity thymidine-³H. The data have been employed to construct a model for repair of UV-induced damage in Drosophila DNA. Implications of the model for DNA repair in mammals are discussed.