Bloom syndrome, characterized by a predisposition to cancer, is caused by mutation of the RecQ DNA helicase gene BLM. The precise function of BLM remains unclear. Previous research suggested that Drosophila BLM functions in the repair of DNA double-strand breaks. Most double-strand breaks in flies are repaired by homologous recombination through the synthesis-dependent strand-annealing pathway. Here, we demonstrate that Drosophila BLM mutants are severely impaired in their ability to carry out repair DNA synthesis during synthesis-dependent strand annealing. Consequently, repair in the mutants is completed by error-prone pathways that create large deletions. These results suggest a model in which BLM maintains genomic stability by promoting efficient repair DNA synthesis and thereby prevents double-strand break repair by less precise pathways.
Although in Saccharomyces cerevisiae the initiation of meiotic recombination, as indicated by double-strand break formation, appears to be functionally linked to the initiation of synapsis, meiotic chromosome synapsis in Drosophila females occurs in the absence of meiotic exchange. Electron microscopy of oocytes from females homozygous for either of two meiotic mutants (mei-W68 and mei-P22), which eliminate both meiotic crossing over and gene conversion, revealed normal synaptonemal complex formation. Thus, synapsis in Drosophila is independent of meiotic recombination, consistent with a model in which synapsis is required for the initiation of meiotic recombination. Furthermore, the basic processes of early meiosis may have different functional or temporal relations, or both, in yeast and Drosophila.In the classical view of meiosis, homologous chromosome synapsis, as indicated by the formation of an elaborate ribbonlike structure called the synaptonemal complex (SC), was thought to be the first and primary event of meiotic prophase, essential for the initiation of meiotic recombination (1). Studies in Saccharomyces cerevisiae, however, have created a different view of the meiotic process in which the initiation of recombination, as evidenced by a doublestrand break (DSB), precedes the initiation of synapsis (2, 3). Three lines of evidence support this view of early meiotic prophase in yeast. First, the initiating event of meiotic recombination, the formation of a DSB, appears before SC formation (4). Second, meiotic mutants that either fail to create DSBs or to process DSBs to make single-stranded tails prevent the formation of a mature SC (2). Third, some mutants allow high levels of meiotic recombination but prevent the production of a mature SC (5). These data are consistent with a model in which single-stranded DNA generated by a DSB carries out a homology search required for synapsis and SC formation. In contrast, synapsis is not an absolute prerequisite for either the initiation (6) or completion of meiotic recombination (7).To assess the relation between synapsis and the initiation of recombination in Drosophila oocytes, we examined both recombination and SC formation in oocytes homozygous for either of two null-recombination mutations. The mei-W68 and mei-P22 (8) mutants prevent the initiation of meiotic recombination as defined by four independent assays: (i) reduction or elimination of meiotic gene conversion; (ii) elimination of meiotic crossing over, as assayed by measuring either intragenic crossing over or the frequency of meiotic crossing over along entire chromosome arms; (iii) lack of doublestrand DNA breaks that persist into metaphase or anaphase I; and (iv) failure to produce either early or late recombination nodules (RNs).To assay the effects of the mei-W68 and mei-P22 mutations on meiotic crossing over, we examined intragenic recombination at the rosy locus (9). No gene conversion events or intragenic crossovers were observed among the progeny of mei-W68 or mei-P22 females (Table 1 and Fig...
Summary DNA recombination and repair pathways require structure-specific endonucleases to process DNA structures that include forks, flaps, and Holliday junctions. Previously, we determined that the Drosophila MEI-9-ERCC1 endonuclease interacts with the novel MUS312 protein to produce meiotic crossovers, and that MUS312 has a MEI-9-independent role in interstrand crosslink (ICL) repair. The importance of MUS312 to pathways crucial for maintaining genomic stability in Drosophila prompted us to search for orthologs in other organisms. Based on sequence, expression pattern, conserved protein-protein interactions, and ICL repair function, we determined that the mammalian ortholog of MUS312 is BTBD12. Orthology between these proteins and S. cerevisiae Slx4 helped identify a conserved interaction with a second structure-specific endonuclease, SLX1. Genetic and biochemical evidence described here and in related papers suggest that MUS312 and BTBD12 direct Holliday junction resolution by at least two distinct endonucleases in different recombination and repair contexts.
The D. melanogaster mei-41 gene is required for DNA repair, mitotic chromosome stability, and normal levels of meiotic recombination in oocytes. Here we show that the predicted mei-41 protein is similar in sequence to the ATM (ataxia telangiectasia) protein from humans and to the yeast rad3 and Mec1p proteins. There is also extensive functional overlap between mei-41 and ATM. Like ATM-deficient cells, mei-41 cells are exquisitely sensitive to ionizing radiation and display high levels of mitotic chromosome instability. We also demonstrate that mei-41 cells, like ATM-deficient cells, fail to show an irradiation-induced delay in the entry into mitosis that is characteristic of normal cells. Thus, the mei-41 gene of Drosophila may be considered to be a functional homolog of the human ATM gene.
Bloom Syndrome, a rare human disorder characterized by genomic instability and predisposition to cancer, is caused by mutation of BLM, which encodes a RecQ-family DNA helicase. The Drosophila melanogaster ortholog of BLM, DmBlm, is encoded by mus309. Mutations in mus309 cause hypersensitivity to DNA-damaging agents, female sterility, and defects in repairing double-strand breaks (DSBs). To better understand these phenotypes, we isolated novel mus309 alleles. Mutations that delete the N terminus of DmBlm, but not the helicase domain, have DSB repair defects as severe as those caused by null mutations. We found that female sterility is due to a requirement for DmBlm in early embryonic cell cycles; embryos lacking maternally derived DmBlm have anaphase bridges and other mitotic defects. These defects were less severe for the N-terminal deletion alleles, so we used one of these mutations to assay meiotic recombination. Crossovers were decreased to about half the normal rate, and the remaining crossovers were evenly distributed along the chromosome. We also found that spontaneous mitotic crossovers are increased by several orders of magnitude in mus309 mutants. These results demonstrate that DmBlm functions in multiple cellular contexts to promote genome stability.
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