Most sexual species induce homologous recombination in meiosis via a developmentally programmed pathway that forms numerous DNA double-strand breaks (DSBs) (Keeney, 2007
). Recombination helps homologous chromosomes pair and become physically connected by crossovers, which promote accurate chromosome segregation at Meiosis I. Recombination also alters genome structure by disrupting linkage of sequence polymorphisms on the same DNA molecule (Kauppi et al., 2004
). Thus, meiotic recombination is a powerful determinant of genome diversity and evolution.
Meiotic DSBs are formed by the conserved Spo11 protein, a topoisomerase relative, via a reaction in which a tyrosine severs the DNA backbone and attaches covalently to the 5′ end of the cleaved strand (Keeney, 2007
) (). Two Spo11 molecules work in concert to cut both strands of a duplex. Endonucleolytic cleavage adjacent to the covalent protein-DNA complex liberates Spo11 bound to a short oligonucleotide (oligo) (Neale et al., 2005
). In S. cerevisiae
, there are two major oligo subpopulations differing in length. The longer (mostly ~21–37 nt) and shorter oligos (<12 nt) are equally abundant and may reflect asymmetry of DSB processing (see below). Further resection of 5′ DSB termini yields 3′-single stranded DNA (ssDNA) that is a substrate for strand exchange proteins.
Mapping DSBs by sequencing Spo11 oligos
Recombination is more likely to occur in some genomic regions than others, largely because of nonrandom DSB distributions (Petes, 2001
; Kauppi et al., 2004
). DSBs in S. cerevisiae
show many levels of spatial organization. There are large (tens of kb) DSB-hot and cold domains, within which are short regions, called hotspots, where DSBs form preferentially. Important determinants of this organization include open chromatin structure, presence of certain histone modifications, and, at some loci, binding of sequence-specific transcription factors (TFs) (Petes, 2001
; Lichten, 2008
). However, detailed understanding is lacking of how these and other factors influence DSB locations.
Prior studies of genome-wide DSB distributions used either covalent Spo11-DSB complexes that accumulate in rad50S
-like mutants or ssDNA generated by DSB resection as microarray hybridization probes (e.g., Gerton et al., 2000
; Blitzblau et al., 2007
; Buhler et al., 2007
). These studies provided considerable insight, but had limited quantitative and spatial resolution due to microarray design, dynamic range of hybridization signal, and the large size of DSB-associated DNA used as probes.
We overcame these limitations by using each Spo11 oligo as a tag that records precisely where a break was made. Sequencing these oligos allowed us to quantitatively map DSBs genome-wide at nucleotide resolution, with high sensitivity. This map elucidated chromosome features that govern DSB distributions; allowed us to test longstanding hypotheses concerning influence of TFs, chromatin, and other factors; and uncovered mechanistic details of the formation and early nucleolytic processing of DSBs.