Mutational analysis of the Drosophila DNA repair and recombination gene mei-9

Drosophila mei-9 is essential for several DNA repair and recombination pathways, including nucleotide excision repair (NER), interstrand crosslink repair, and meiotic recombination. To better understand the role of MEI-9 in these processes, we characterized 10 unique mutant alleles of mei-9. These include a P-element insertion that disrupts repair functions but not the meiotic function; three nonsense mutations, one of which has nearly wild-type levels of protein; three missense mutations, one of which disrupts the meiotic function but not repair functions; two small in-frame deletions; and one frameshift.     

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.     

The type of recombination in Drosophila melanogaster mei-9LI males induced by space flight factors

Spontaneous male recombination in mei-9LI stock of Drosophila melanogaster having defect in excision repair was shown to take place at early premeiotic stage. Male recombination was registered at all stages of gametogenesis, under the action of acceleration (6-8 g). The frequencies of male recombination at pre- and postmeiotic stages of gametogenesis increased significantly under the effect of space flight factors.     

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.     

Effect of gamma-irradiation on oogenesis of Drosophila mutants defective for reparation and meiotic recombination

Effects of mutations rad201, mei-9, and mei-41 on cell sensitivity to gamma-radiation in Drosophila oogenesis were studied. Females of the control (Oregon R) and mutant strains were irradiated at a dose of 15 Gy. For 9 days after the irradiation, the number of eggs in consecutive day batches, the frequency of dominant lethals (DLs) among the eggs, and the cytologically recorded distribution of oocytes for stages of their development, and the frequency of egg chamber degeneration in female ovaries were estimated. As a result of joint analysis of the data, different oogenesis stages were characterized with regard to the frequency of two radiation-induced events: appearance of DLs in oocytes and degeneration of egg chambers due to apoptosis of nurse cells. It was shown that the mutations affect these parameters only at particular stages of early oogenesis, at which previtellogenetic growth of egg follicles and meiotic recombination in oocytes occur. Mutation rad201G1 increased the frequency of DLs and egg chamber degeneration, mei-41D5 affected only the DL frequency, and mei-9a, in addition to enhancing the chamber degeneration frequency, promoted radiation "rescue" of some oocytes from the DL induction.     

Unique invasions and resolutions: DNA repair proteins in meiotic recombination in Drosophila melanogaster

To ensure the accurate disjunction of homologous chromosomes during meiosis, most eukaryotes rely on physical connections called chiasmata, which form at sites of crossing over. In the absence of crossing over, homologs may segregate randomly, resulting in high frequencies of aneuploid gametes. The process of meiotic recombination poses unique problems for the cell that must be overcome to ensure normal disjunction of homologous chromosomes. How is it ensured that crossovers occur between homologous chromosomes, rather than between sister chromatids? What determines the number and location of crossovers? The functions of DNA repair proteins hold some of the answers to these questions. In this review, we discuss DNA repair proteins that function in meiotic recombination in Drosophila melanogaster. We emphasize the processes of strand invasion and Holliday junction resolution in order to shed light on the questions raised above. Also, we compare the variety of ways several eukaryotes perform these processes and the different proteins they require.     

Recovery and characterization of hybrid dysgenesis-induced mei-9 and mei-41 alleles of Drosophila melanogaster

X-Linked methyl methanesulfonate (MMS)-sensitive mutations were induced with hybrid dysgenesis using four P strains: pi 2, Harwich, T-007 and OK-1. Mutations were identified after two generations of backcrosses to M strain females to replace the autosomes. Among 51,471 X-chromosomes examined 10 carried stable MMS-sensitive mutations representing 8 independent events. Males of the mutant strains failed to induce gonadal dysgenesis in crosses to Oregon-R females at 28.5 degrees C. Complementation tests showed that 3 of the induced mutations were mei-9 alleles, 2 were mei-41 alleles, 1 was a mus102 allele, and 2 were alleles at a newly identified MMS-sensitive locus, mus112 (map position: 1-32.8). As assayed by in situ hybridization on polytene chromosomes, each X-chromosome had no more than four P element insertions. 4 of the 8 mutations recovered in this study proved to have P element insertions at or very close to sites to which MMS sensitivity has been mapped. Hybrid dysgenesis-induced reversion of 2 mutants, mei-9RT1 and mei-41RT2, is associated with the loss of the P element from regions 4B and 14C respectively.     

A system selective for yeast mutants deficient in meiotic recombination

Summary An experimental design and rationale for detecting and recovering Saccharomyces cerevisiae mutants specifically blocked in meiotic gene conversion is presented. The system utilizes an otherwise haploid strain disomic (n+1) for chromosome III which is simultaneously heterozygous for the mating-type locus and heteroallelic at leu2. The former is an essential requirement for inducing meiotic development; i.e., DNA replication and sporulation upon transfer to acetate media, while the latter provides a convenient signal for assaying recombination at the intragenic level. Of 940 clones screened qualitatively after mutagenesis with ethyl methanesulfonate, 91 presumptive mutants were isolated. These are classed arbitrarily into four groups according to the reduction in interallelic recombination observed in quantitative tests.Supported by research grants GM: 17317 and GM: 16522 from the National Institutes of Health and a grant from the National Sciences Foundation, GB: 8534.     

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.     

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.     

A germline clone screen for meiotic mutants in Drosophila melanogaster

Using an FLP/FRT-based method to create germline clones, we screened Drosophila chromosome arms 2L and 3R for new female meiotic mutants. The screen was designed to recover mutants with severe effects on meiotic exchange and/or segregation. This screen yielded 11 new mutants, including six alleles of previously known meiotic genes (c(2)M and ald/mps1). The remaining five mutants appear to define at least four new genes whose ablation results in severe meiotic defects. Three of the novel meiotic mutants were identified at the molecular level. Two of these, mcm5(A7) and trem(F9), define roles in meiotic recombination, while a third, cona(A12), is important for synaptonemal complex assembly. Surprisingly, five of the nine mutants for which the lesion has been identified at the molecular level are not the result of mutations characteristic of EMS mutagenesis, but rather due to the insertion of the transposable element Doc. This study demonstrates the utility of germline clone-based screens for the discovery of strong meiotic mutants, including mutations in essential genes, and the use of molecular genetic techniques to map the loci.     

Apurinic endonuclease activity from wild-type and repair-deficient mei-9 Drosophila ovaries.

Summary An endonuclease which acts on apurinic (AP) sites in DNA was partially purified from Drosophila ovaries. The enzyme present in the female germ line has a molecular weight of 63 000 daltons, is Mg⁺⁺ dependent, and produces a site upon cleaving depurinated DNA that supports DNA repair synthesis. Although the same characteristics are shared by the enzyme present in the excision-deficient mutant mei-9, specific activity for the AP endonuclease is reduced 98% when compared with that found for its wild-type counterpart. Moreover, cross-reactivity toward an antibody that recognizes the wild-type AP endonuclease protein is reduced roughly 90% for partially purified preparations from mei-9. Mixing experiments between extracts of mei-9 and wild type suggest that the mei-9 structural gene somehow alters or influences the levels of the AP endonuclease protein, but in view of the complex phenotype of this mutant the endonuclease is probably not the product of the gene itself.     

Intermediates of yeast meiotic recombination contain heteroduplex DNA

The formation of heteroduplex DNA features prominently in all models for homologous recombination. A central intermediate in the current double-strand break repair model contains two Holliday junctions flanking a region of heteroduplex DNA. Studies of yeast meiosis have identified such intermediates but failed to detect associated heteroduplex DNA. We show here that these intermediates contain heteroduplex DNA, providing an important validation of the double-strand break repair model. However, we also detect intermediates where both Holliday junctions are to one side of the initiating DSB site, while the intervening region shows no evidence of heteroduplex DNA. Such structures are not easily accommodated by the canonical version of the double-strand break repair model.     

Yeast meiotic mutants proficient for the induction of ectopic recombination.

A screen was designed to identify Saccharomyces cerevisiae mutants that were defective in meiosis yet proficient for meiotic ectopic recombination in the return-to-growth protocol. Seven mutants alleles were isolated; two are important for chromosome synapsis (RED1, MEK1) and five function independently of recombination (SPO14, GSG1, SPOT8/MUM2, 3, 4). Similar to the spoT8-1 mutant, mum2 deletion strains do not undergo premeiotic DNA synthesis, arrest prior to the first meiotic division and fail to sporulate. Surprisingly, although DNA replication does not occur, mum2 mutants are induced for high levels of ectopic recombination. gsg1 diploids are reduced in their ability to complete premeiotic DNA synthesis and the meiotic divisions, and a small percentage of cells produce spores. mum3 mutants sporulate poorly and the spores produced are inviable. Finally, mum4-1 mutants produce inviable spores. The meiotic/sporulation defects of gsg1, mum2, and mum3 are not relieved by spo11 or spo13 mutations, indicating that the mutant defects are not dependent on the initiation of recombination or completion of both meiotic divisions. In contrast, the spore inviability of the mum4-1 mutant is rescued by the spo13 mutation. The mum4-1 spo13 mutant undergoes a single, predominantly equational division, suggesting that MUM4 functions at or prior to the first meiotic division. Although recombination is variably affected in the gsg1 and mum mutants, we hypothesize that these mutants define genes important for aspects of meiosis not directly related to recombination.     

Mutant alleles of the meiotic locus, mei-9, differ in degree of effects on rod chromosome magnification and ring chromosome transmission in Drosophila

Two mutant alleles of the meiotic locus, mei-9, have been examined for their effect on magnification of a rod Xbb chromosome and transmission of a ring Xbb chromosome under magnifying conditions. Our results indicate that the effects of these two mutations are allele-specific: mei-9a strongly inhibits both rod chromosome magnification and ring chromosome loss under magnifying conditions, while mei-9b has a smaller inhibitory effect on rod chromosome magnification and on the transmission of ring chromosomes under magnifying conditions. These observations can be explained by a difference in leakiness between the two alleles. Our results demonstrate that mutants defective in excision repair and repair replication inhibit ribosomal gene magnification. This suggests that a component of the excision repair pathway is involved in the process of magnification.     

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.     

PRDM9 points the zinc finger at meiotic recombination hotspots

Meiotic recombination events are spread nonrandomly across eukaryotic genomes in 'hotspots'. Recent work shows that a unique histone methyltransferase, PRDM9, determines their distribution.