Abstract
DNA double-strand breaks (DSBs), which are formed by the Spo11 protein, initiate meiotic recombination. Previous DSB-mapping studies have used rad50S or sae2Δ mutants, which are defective in break processing, to accumulate Spo11-linked DSBs, and report large (≥ 50 kb) “DSB-hot” regions that are separated by “DSB-cold” domains of similar size. Substantial recombination occurs in some DSB-cold regions, suggesting that DSB patterns are not normal in rad50S or sae2Δ mutants. We therefore developed a novel method to map genome-wide, single-strand DNA (ssDNA)–associated DSBs that accumulate in processing-capable, repair-defective dmc1Δ and dmc1Δ rad51Δ mutants. DSBs were observed at known hot spots, but also in most previously identified “DSB-cold” regions, including near centromeres and telomeres. Although approximately 40% of the genome is DSB-cold in rad50S mutants, analysis of meiotic ssDNA from dmc1Δ shows that most of these regions have substantial DSB activity. Southern blot assays of DSBs in selected regions in dmc1Δ, rad50S, and wild-type cells confirm these findings. Thus, DSBs are distributed much more uniformly than was previously believed. Comparisons of DSB signals in dmc1, dmc1 rad51, and dmc1 spo11 mutant strains identify Dmc1 as a critical strand-exchange activity genome-wide, and confirm previous conclusions that Spo11-induced lesions initiate all meiotic recombination.
Highlights
Meiosis results in the faithful and efficient division of a diploid genome into four haploid gametes
DNA double-strand breaks (DSBs), which are formed by the conserved Spo11 nuclease, initiate meiotic recombination
We developed a new DSB mapping method that purifies and analyzes the single-strand DNA formed at breaks after Spo11 removal
Summary
Meiosis results in the faithful and efficient division of a diploid genome into four haploid gametes. Meiotic recombination promotes genetic diversity, but its main role is to ensure interhomolog association during the first meiotic division [1] This association is absolutely required for efficient homolog separation, and defects in meiotic recombination result in chromosome nondisjunction [2]. Subsequent to DSB formation, Spo is removed by endonucleolytic cleavage [13], and break ends undergo 59 to 39 resection to create 39 end single-strand tails [14]. This produces a substrate for Dmc and Rad, which are eukaryotic RecA homologues that catalyze the strandinvasion step of meiotic DSB repair by interhomolog recombination [15,16]. Dmc is expressed only during meiosis and is responsible for the bulk of meiotic DSB repair, whereas Rad is required for homologous recombination during vegetative growth and contributes to meiotic recombination [17,18]
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