Mapping Meiotic Single-Strand DNA Reveals a New Landscape of DNA Double-Strand Breaks in Saccharomyces cerevisiae

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.

Rec8 Guides Canonical Spo11 Distribution along Yeast Meiotic Chromosomes

“…In the mutant, Spo11 is removed from the DSB ends, but unrepaired single-strand DNA accumulates (Bishop et al, 1992). Similar distribution of DSB was observed in both types of mutants except for some regions: in the dmc1⌬ mutant, DSBs were formed also in the DSB cold domains located near centromeres and chromosome ends in the rad50S like mutant (Blitzblau et al, 2007;Buhler et al, 2007).…”

“…At the 1.5 h after meiotic induction, when premeiotic DNA replication is about to start (see Figure 1A and Supplemental Figure S4), initial binding of Spo11-FLAG on chromosome VI was detected mainly within the 20-to 30-kb regions harboring the centromere (referred to as pericentromeric regions in this study) and at some scattered sites on the chromosome arms ( Figure 1B). The ChIP-chip experiments for Rec8-FLAG demonstrated that Rec8 also initially bound pericentromeric regions at the DSBs formation has not been detected at inside centromeres, and it is likely to be reduced in 8-to 10-kb regions from centromeres in S. cerevisiae (Blitzblau et al, 2007;Buhler et al, 2007). To validate the binding of Spo11-FLAG to the pericentromeric regions, we performed quantitative PCRbased ChIP experiments (Figure 2, B-D).…”

“…For example, chromatin remodeling factors and histone modifications such as acetylation, methylation, and ubiquitination are involved in the meiotic alteration of the local chromatin structure at DSB sites (Sollier et al, 2004;Yamada et al, 2004;Yamashita et al, 2004;Mieczkowski et al, 2007). In addition, genome-wide studies have shown that DSB regions are distributed nonrandomly in DSB hot and cold domains (Baudat and Nicolas, 1997;Gerton et al, 2000;Borde et al, 2004;Blitzblau et al, 2007;Buhler et al, 2007) and that DSBs are formed preferentially in regions of chromatin loops (Blat et al, 2002).…”

“…The binding sites of Spo11 in rad50S-like mutants have been considered as DSB formation sites. Recently, DSB sties were genome-widely mapped by detecting single-strand DNA sites in the deletion mutant of DMC1, a eukaryotic RecA homologue, which catalyzes the single-strand invasion (Blitzblau et al, 2007;Buhler et al, 2007). In the mutant, Spo11 is removed from the DSB ends, but unrepaired single-strand DNA accumulates (Bishop et al, 1992).…”

The distribution of alternating AT sequences in eukaryotic genomes suggests a role in homologous chromosome recognition in meiosis

“…They could form complexes with other proteins known to play an essential role in meiotic chromosome pairing in yeast, such as SPO11 [36], MER3 [37], RAD51, the MRX complex [38], etc. However it should be mentioned that these and other known proteins, as reviewed by Hunter [38], only play a role in meiotic recombination after the process of homologue recognition has taken place.…”