mus309 mutation, defective in DNA double-strand break repair, affects intergenic but not intragenic meiotic recombination in Drosophila melanogaster

The effect was investigated of the hypomorphic DNA double-strand break repair, notably synthesis-dependent strand annealing, deficient mutation mus309 on the third chromosome of Drosophila melanogaster on intergenic and intragenic meiotic recombination in the X chromosome. The results showed that the mutation significantly increases the frequency of intergenic crossing over in two of three gene intervals of the X chromosome studied. Interestingly the increase was most prevalent in the tip of the X chromosome where crossovers normally are least frequent per physical map unit length. In particular crossing over interference was also affected, indicating that the effect of the mus309 mutation involves preconditions of crossing over but not the event of crossing over itself. On the other hand, the results also show that most probably the mutation does not have any effect on intragenic recombination, i.e. gene conversion. These results are fully consistent with the present molecular models of meiotic crossing over initiated by double-strand breaks of DNA followed by formation of a single-end-invasion intermediate, or D-loop, which is subsequently processed to generate either crossover or non-crossover products involving formation of a double Holliday junction. In particular the results suggest that the mus309 gene is involved in resolution of the D-loop, thereby affecting the choice between double-strand-break repair (DSBR) and synthesis-dependent strand annealing (SDSA) pathways of meiotic recombination.     

Evaluation of effects of gamma-irradiation at low doses on repair and meiotic recombination mutants of Drosophila melanogaster

A comparative study of the effects of gene mutations mus209, mus309, mei-41 and rad54 of Drosophila melanogaster on the sensitivity to low-level exposure of different duration was carried out. Taken into account was the survival rate at different stages of ontogeny, female fecundity, the frequency of dominant lethal mutations (DLM) and the DNA damage. mei-41 and rad-54 mutants were most sensitive to the action of low dose radiation (80 mGy) in terms of survival and DLM. However, at the level of DNA damage, an increased radiosensitivity is observed only at larger doses of low intensity irradiation. Based on these observations, we can conclude about the importance of repair and its genes in the formation of the effect of low level doses of ionizing radiation in Drosophila.     

Drosophila brca2 is required for mitotic and meiotic DNA repair and efficient activation of the meiotic recombination checkpoint

Heterozygous mutations in the tumor suppressor BRCA2 confer a high risk of breast and other cancers in humans. BRCA2 maintains genome stability in part through the regulation of Rad51-dependent homologous recombination. Much about its precise function in the DNA damage responses is, however, not yet known. We have made null mutations in the Drosophila homolog of BRCA2 and measured the levels of homologous recombination, non-homologous end-joining, and single-strand annealing in the pre-meiotic germline of Drosophila males. We show that repair by homologous recombination is dramatically decreased in Drosophila brca2 mutants. Instead, large flanking deletions are formed, and repair by the non-conservative single-strand annealing pathway predominates. We further show that during meiosis, Drosophila Brca2 has a dual role in the repair of meiotic double-stranded breaks and the efficient activation of the meiotic recombination checkpoint. The eggshell patterning defects that result from activation of the meiotic recombination checkpoint in other meiotic DNA repair mutants can be strongly suppressed by mutations in brca2. In addition, Brca2 co-immunoprecipitates with the checkpoint protein Rad9, suggesting a direct role for Brca2 in the transduction of the meiotic recombination checkpoint signal.     

X-ray quality and the induction of meiotic recombination in Drosophila melanogaster males.

This paper reports a set of experiments designed to determine whether the radiation-quality effect, reported for the induction of somatic recombination, could be also demonstrated for meiotic recombination. Males heterozygous for markers on the 2nd chromosome were given an exposure of 600 R with either 55 kV or 100 kV X-irradiation. Treated spermatocytes were sampled by irradiating young pupae and then taking the first day sperm from the emergent adult males. When 2-h old pupae were irradiated there were significantly fewer centric and more non-centric recombinants, than when 4--6 h old pupae were irradiated. No radiation-quality effect was found.     

Heteroduplex DNA in meiotic recombination in Drosophila mei-9 mutants

Meiotic recombination gives rise to crossovers, which are required in most organisms for the faithful segregation of homologous chromosomes during meiotic cell division. Characterization of crossover-defective mutants has contributed much to our understanding of the molecular mechanism of crossover formation. We report here a molecular analysis of recombination in a Drosophila melanogaster crossover-defective mutant, mei-9. In the absence of mei-9 activity, postmeiotic segregation associated with noncrossovers occurs at the expense of crossover products, suggesting that the underlying meiotic function for MEI-9 is in crossover formation rather than mismatch repair. In support of this, analysis of the arrangement of heteroduplex DNA in the postmeiotic segregation products reveals different patterns from those observed in Drosophila Msh6 mutants, which are mismatch-repair defective. This analysis also provides evidence that the double-strand break repair model applies to meiotic recombination in Drosophila. Our results support a model in which MEI-9 nicks Holliday junctions to generate crossovers during meiotic recombination, and, in the absence of MEI-9 activity, the double Holliday junction intermediate instead undergoes dissolution to generate noncrossover products in which heteroduplex is unrepaired.     

Inhibitors of thymidylate synthesis increase whereas thymidine decreases meiotic recombination in Drosophila melanogaster

Summary We have demonstrated the effect of different media on meiotic recombination in Drosophila melanogaster. Recombination is more frequent when the medium is deprived of bases, nucleosides and nucleotides. We have shown that two inhibitors of thymidylate (dTMP) synthesis —aminopterin inhibiting dihydrofolate reductase (DHFR) and fluorodeoxyuridine (FUdR) inhibiting thymidylate synthetase-result in a significant increase in meiotic recombination in the yellow/white region on the X chromosome of Drosophila melanogaster. Moreover the addition of thymidine to the richest medium significantly lowers normal recombination. Such studies represent a powerful tool for future studies on the mechanism of meiotic recombination.     

Enzymatic studies of DNA repair in Drosophila melanogaster

Thus far, our studies in Drosophila have concentrated primarily on the various enzymes involved in the in vitro repair of modified or nonconventional DNA substrates. In some cases, our findings have led us to investigate events that may not have a bearing on DNA repair, but rather may be associated with developmental signals important to the maturation of the organism. As appealing as some of these models seem, however, they must await confirmation through detailed genetic studies before any substantial conclusions can be drawn. This combination of genetic and biochemical knowledge makes Drosophila an exciting organism for an eventual detailed understanding of the developmental expression and cellular location of DNA-repair systems.     

Selection and recombination in Drosophila melanogaster

Summary Artificial selection for wing length in Drosophila melanogaster resulted in changed crossing-over frequencies between three marker genes on the 2nd chromosome, b, cn and vg.The results suggest that artificial selection is a causal agent in producing the observed changes; moreover it is suggested that the modifications in cross-over frequency are controlled by extra-nuclear factors.Research supported by C.N.R. Consiglio Nazionale delle Ricerche Roma, Grant n. 115.2298.4791.     

The effects of recombination-defective meiotic mutants in Drosophila melanogaster on gonial recombination in males.

Recombination-defective female meiotic mutants representing 7 loci in Drosophila melanogaster have been examined for effects on gonial recombination in males. These loci were chosen for study because they represent a broad range of the known types of defects in processes necessary for meiotic recombination and somatic chromosome stability. Alleles at 6 of the loci studied did not increase the frequency of gonial recombination in males, whereas a mutant at one locus was associated with an increase (about 10-fold) in gonial recombination. These results suggest that the defects in chromosomal metabolism caused by these recombination, and in some cases repair, defective mutants are distinct from those of the male-recombination promoting elements (Mr) recently isolated from many natural populations. Analysis of the spontaneous events detected in this study showed that a third to a half of the events detected are actually of mutational rather than recombinational origin.     

Functional analysis of Drosophila melanogaster BRCA2 in DNA repair

The human BRCA2 cancer susceptibility protein functions in double-strand DNA break repair by homologous recombination and this pathway is conserved in the fly Drosophila melanogaster. Although a potential Drosophila melanogaster BRCA2 orthologue (dmbrca2; CG30169) has been identified by sequence similarity, no functional data addressing the role of this protein in DNA repair is available. Here, we demonstrate that depletion of dmbrca2 from Drosophila cells induces sensitivity to DNA damage induced by irradiation or treatment with hydroxyurea. Dmbrca2 physically interacts with dmrad51 (spnA) and the two proteins become recruited to nuclear foci after DNA damage. A functional assay for DNA repair demonstrated that in flies dmbrca2 plays a role in double-strand break repair by gene conversion. Finally, we show that depletion of dmbrca2 in cells is synthetically lethal with deficiency in other DNA repair proteins including dmparp. The conservation of the function of BRCA2 in Drosophila will allow the analysis of this key DNA repair protein in a genetically tractable organism potentially illuminating mechanisms of carcinogenesis and aiding the development of therapeutic agents.     

Role of ubiquitination in meiotic recombination repair

Programmed and unprogrammed double-strand breaks (DSBs) often arise from such physiological requirements as meiotic recombination, and exogenous insults, such as ionizing radiation (IR). Due to deleterious impacts on genome stability, DSBs must be appropriately processed and repaired in a regulatory manner. Recent investigations have indicated that ubiquitination is a critical factor in DNA damage response and meiotic recombination repair. This review summarizes the effects of proteins and complexes associa...     

mei-P22 encodes a chromosome-associated protein required for the initiation of meiotic recombination in Drosophila melanogaster

Double-strand breaks (DSB) initiate meiotic recombination in a variety of organisms. Here we present genetic evidence that the mei-P22 gene is required for the induction of DSBs during meiotic prophase in Drosophila females. Strong mei-P22 mutations eliminate meiotic crossing over and suppress the sterility of DSB repair-defective mutants. Interestingly, crossing over in mei-P22 mutants can be restored to almost 50% of wild-type by X irradiation. In addition, an antibody-based assay was used to demonstrate that DSBs are not formed in mei-P22 mutants. This array of phenotypes is identical to that of mei-W68 mutants; mei-W68 encodes the Drosophila Spo11 homolog that is proposed to be an enzyme required for DSB formation. Consistent with a direct role in DSB formation, mei-P22 encodes a basic 35.7-kD protein, which, when examined by immunofluorescence, localizes to foci on meiotic chromosomes. MEI-P22 foci appear transiently in early meiotic prophase, which is when meiotic recombination is believed to initiate. By using an antibody to C(3)G as a marker for synaptonemal complex (SC) formation, we observed that SC is present before MEI-P22 associates with the chromosomes, thus providing direct evidence that the development of SC precedes the initiation of meiotic recombination. Similarly, we found that MEI-P22 foci did not appear in a c(3)G mutant in which SC does not form, suggesting that DSB formation is dependent on SC formation in Drosophila. We propose that MEI-P22 interacts with meiosis-specific chromosome proteins to facilitate DSB creation by MEI-W68.     

Temporal analysis of meiotic DNA double-strand break formation and repair in Drosophila females

Using an antibody against the phosphorylated form of His2Av (γ-His2Av), we have described the time course for the series of events leading from the formation of a double-strand break (DSB) to a crossover in Drosophila female meiotic prophase. MEI-P22 is required for DSB formation and localizes to chromosomes prior to γ-His2Av foci. Drosophila females, however, are among the group of organisms where synaptonemal complex (SC) formation is not dependent on DSBs. In the absence of two SC proteins, C(3)G and C(2)M, the number of DSBs in oocytes is significantly reduced. This is consistent with the appearance of SC protein staining prior to γ-His2Av foci. However, SC formation is incomplete or absent in the neighboring nurse cells, and γ-His2Av foci appear with the same kinetics as in oocytes and do not depend on SC proteins. Thus, competence for DSB formation in nurse cells occurs with a specific timing that is independent of the SC, whereas in the oocytes, some SC proteins may have a regulatory role to counteract the effects of a negative regulator of DSB formation. The SC is not sufficient for DSB formation, however, since DSBs were absent from the heterochromatin even though SC formation occurs in these regions. All γ-His2Av foci disappear before the end of prophase, presumably as repair is completed and crossovers are formed. However, oocytes in early prophase exhibit a slower response to X-ray–induced DSBs compared to those in the late pachytene stage. Assuming all DSBs appear as γ-His2Av foci, there is at least a 3:1 ratio of noncrossover to crossover products. From a comparison of the frequency of γ-His2Av foci and crossovers, it appears that Drosophila females have only a weak mechanism to ensure a crossover in the presence of a low number of DSBs.     

Non-homologue pairing and spontaneous meiotic interchanges in Drosophila melanogaster females

Spontaneous interchange between the X chromosomes and the C(2L) autosomal compound in their centromeric regions was studied in y/XY;C(2L);C(2R) and In(1)dl-49+BM1/XY;C(2L);C(2R) Drosophila melanogaster females. These females were mated with F(2L)/F(2L);C(2R) males. Interchange occurrence was recorded as the appearance of an F1 individual with a half-translocation of either X . 2L or Y . 2L type. 37 interchanges were recovered in y/XY and 67 in In(1)/XY females. The majority of the interchanges were of meiotic origin. The interchanges were mainly C(2L)-XY; the most frequent type of half-translocation was Y . 2L;dl-49+BM1. Inversion increased about 5-fold the interchange frequency. In the course of C(2L)-XY interchange, the other X chromosome and C(2R) compound regularly paired and disjoined. In y/XY females, 8 crossover half-translocations of meiotic origin were recovered. The results obtained indicate that meiotic pairing between the X's and C(2L) occurred in the females examined. According to our estimates, XY-C(2L) pairing is associated with interchange in the heterochromatic centromeric regions with a frequency of 10(-3). The recovery of crossover half-translocations supports the chromocentral model of non-homologous pairing and allows us to assume that a chromosome may simultaneously pair with a homologue and a non-homologue. The disjunction pattern of this trivalent depends on its structure in each particular case. The chromosome-segregation pattern resulting from spontaneous interchanges was similar to that resulting from radiation-induced interchanges in the immature oocytes described by Parker. This similarity suggests that non-homologue pairing occurs in the immature oocytes too. The non-homologue-pairing pattern established by the interchange test conformed well with that previously established in y/XY and In(1)XY females by the distribution test.