Protein Determinants of Meiotic DNA Break Hot Spots

Abstract

SUMMARY Meiotic recombination, crucial for proper chromosome segregation and genome evolution, is initiated by programmed DNA double-strand breaks (DSBs) in yeasts and likely all sexually reproducing species. In fission yeast, DSBs occur up to hundreds of times more frequently at special sites, called hotspots, than in other regions of the genome. What distinguishes hotspots from cold regions is an unsolved problem, although transcription factors determine some hotspots. We report the discovery that three coiled-coil proteins – Rec25, Rec27, and Mug20 – bind essentially all hotspots with unprecedented specificity even without DSB formation. These small proteins are components of linear elements, are related to synaptonemal complex proteins, and are essential for nearly all DSBs at most hotspots. Our results indicate these hotspot determinants activate or stabilize the DSB-forming protein Rec12 (Spo11 homolog) rather than promote its binding to hotspots. We propose a new paradigm for hotspot determination and crossover control by linear element proteins.

Evolutionarily diverse determinants of meiotic DNA break and recombination landscapes across the genome

“…We infer that many of the longer multi-mappers reflect Rec12 oligos from truly repetitive DNA elements (e.g., rDNA, pericentric repeats, and transposable elements), whereas most of the shorter reads, although mapped ambiguously, came primarily from nonrepetitive genomic regions (i.e., repeated sequences longer than the oligo read but less than 50 bp long). In support of this conclusion, multimappers were enriched in previously defined DSB hotspots (Fowler et al 2013) in proportion to the unique oligo map (Supplemental Fig. S1C), and multi-mappers and unique reads showed similar fine-scale distributions within hotspots (Supplemental Fig.…”

“…(E) Quantitative correlation between Rec12 oligos and Rec12 ChIP-chip. Rec12 oligos were summed at hotspots determined by ChIP-chip (n = 288) and compared with the integrated microarray signal (Fowler et al 2013). The trend is slightly nonlinear, likely because of higher microarray background.…”

“…Three S. pombe proteins (Rec25, Rec27, and Mug20), likely acting as a complex, bind essentially all hotspots and are required for most DSB formation there, but how they act and the chromosome features determining their binding remain unknown (Fowler et al 2013). In a few cases, hotspots depend on transcription factors (TFs) binding a particular DNA sequence, e.g., at the ade6-M26 mutation in S. pombe and the HIS4 locus in S. cerevisiae (Schuchert et al 1991;White et al 1991;Kon et al 1997;Steiner et al 2002).…”

“…As a result, S. pombe has fewer, more widely spaced hotspots than S. cerevisiae. We previously defined 288 DSB hotspots as regions with ChIP-chip hybridization signal at or above the level expected for a DSB frequency of 0.3%, based on linear regression of ChIPchip signal vs. direct DSB Southern blot assays at 25 hotspots (Cromie et al 2007;Fowler et al 2013). This value (0.3%) represents the approximate threshold for detection on the Southern blots (Cromie et al 2007).…”

“…The density in the weakest hotspot scored was thus 247-fold higher than the density in the rDNA, and 2.2-fold higher than the genome average. This list accounted for 98% of the 288 previously defined hotspots (Fowler et al 2013), plus 322 additional hotspots uncovered because of the greater sensitivity of Rec12-oligo sequencing. Consistent with previous findings, most Rec12-oligo hotspots overlapped with IGRs ( Fig.…”