GSE171042
Spo11 generates gaps through concerted cuts at sites of topological stress [dDSB]
Meiotic recombination is essential for proper meiotic chromosome segregation and fertility, and is initiated by programmed DNA double-strand breaks (DSBs) introduced by Spo11, a eukaryotic homolog of archaeal topoisomerase VIA. Here we report the discovery of hitherto uncharacterized Spo11-induced lesions, small gaps from 34 bp to several hundred bp, which are generated by coordinated pairs of DSBs (double DSBs or dDSBs). Isolation and genome-wide mapping of the resulting fragments with single base pair precision reveals enrichment at DSB hotspots but also a widely dispersed distribution covering the entire genome. We show that Spo11 prefers to cut at a sequence motif which promotes DNA bending, indicating that bendability of DNA contributes to cleavage site choice. Moreover, fragment lengths display a ~ (10.4n+3) bp periodicity, implying that Spo11 favours cleavage on the same face of underwound DNA. Consistently, dDSB signals overlap and correlate with topoisomerase II binding sites, which points to a role for topological stress and DNA crossings in break formation, and suggests a unified model for DSB and dDSB formation, in which Spo11 traps two DNA strands. Furthermore, gaps resulting from dDSBs, an estimated 20% of all initiation events, can account for full gene conversion events that are independent of both Msh2-dependent heteroduplex repair and MutL?. Since non-homologous gap repair results in deletions, and ectopically re-integrated dDSB fragments result in insertions, dDSB formation represents a potential source of evolutionary diversity and pathogenic germ-line aberrations. Overall design: dDSB fragments from specific time points of meiotic yeast cultures were isolated via tagged Spo11 by Protec-Seq and ligated to Illumina adapters for amplification and (single or) paired-end 50 bp or 75 bp sequencing on Illumina platforms. Saccharomyces cerevisiae(S. cer.) cells were mixed with the same batch of Saccharomyces kudriavzevii (S. kud.) cells for downstream quantitative comparison. We are providing 31 immunoprecipitation (IP) and 31 corresponding whole cell extract (WCE) samples from wild-type and various mutant S. cer. strains, as well as one S. kud. sample.