the whole genome (median of 36.6% versus 36.0%; p < 0.0001 by two-tailed t-test). However, base composition is a very poor predictor of prominent Rec12 peak position, as 37% of prominent Rec12 peaks are centered on windows with GC content below the global median and only 0.2% of windows with GC content above the global median are centered on a prominent Rec12 peak. No significant GC content enrichment was seen at weak Rec12 peak positions. Sliding windows of 0.5 kb, 1 kb, or 10 kb showed even less difference between peak-centered and whole-genome window compositions (unpublished data). In S. cerevisiae, DSB hotspots are also weakly correlated with higher than average GC content when a 5-kb sliding window is used .
Comparison of S. pombe, S. cerevisiae, and Human DSB Hotspot Distributions
As in S. pombe, DSBs have been analyzed in S. cerevisiae by both direct Southern blot analysis and microarray analysis of DNA covalently linked to Spo11, the Rec12 homolog [e.g.,20,21,35]. These analyses show that DSB hotspots in S. cerevisiae are in general slightly less prominent and often more closely spaced than those in S. pombe. For example, on the most thoroughly analyzed S. cerevisiae Chromosome III, the maximal fraction of DNA broken at a hotspot is 9% , and the maximal enrichment of Spo11-DNA linkage is 8-fold  or 15-fold . In many regions of the genome, most of the open reading frames have a detectable DSB at their 5′ end.
In S. pombe many DSB hotspots have 10%–15% of the DNA broken (Figures 4 and S6) , and Rec12-enrichment factors (IP:WCE ratio) are often >25 and occasionally up to 50 (Figures 3 and S7). Between the prominent Rec12 peaks, there are frequent intervals of 50–100 kb with few if any DSBs detectable by either Southern blot or microarray analysis (Figures 3 and S7) . We have not noted continuous stretches of genes with DSB hotspots at their 5′ ends. Thus, DSB hotspots (or small groups of hotspots) are usually isolated from others in S. pombe. In spite of hotspots being far apart, crossovers in S. pombe are nearly uniformly distributed . Crossing-over far from detectable DSBs may involve migration of DNA intermediates far from a DSB, or they may be initiated by DNA lesions not detectable by current technology [12,36].
In these two yeasts meiotic DSBs are distributed differently in the centromeric and telomeric regions than in other regions of the genome. In S. cerevisiae centromeres are small (∼0.1 kb), and DSBs are infrequent to one or both sides of many of the centromeres [10,20,21,35]. In S. pombe centromeres are large (35–100 kb), and DSBs are undetectable within them by microarray analysis (Figure S7) or by Southern blot analysis (C. Ellermeier and G. Smith, unpublished data). In the adjacent intervals, however, DSBs appear as frequent as in other regions of the genome. In both yeasts ChIP-on-chip analysis suggests that Rec12 (Spo11)-DNA linkages are under-represented in the terminal 40 kb by ∼20% (unpublished data) [10,20,21]. In S. pombe, however, there is a prominent DSB ∼18 kb from the left end of Chromosome I, including ∼10 kb of unsequenced DNA (Figure 3), although prominent Rec12 peaks occur only 70–150 kb from the other three telomeres. DSB-free intervals of similar size occur throughout the chromosomes. Thus, with respect to meiotic DSBs S. pombe telomeres do not appear substantially different from other regions of the genome.
Meiotic DSBs have not been directly detected in human cells, but hotspots of recombination, presumably reflecting hotspots of DSB formation, have been detected using both linkage disequilibrium analysis and sperm typing. These methods have detected small regions of DNA, often only 1–2 kb wide by sperm typing, where meiotic recombination rates are greatly elevated compared to neighboring intervals or the genome average [37–40]. Like S. pombe and S. cerevisiae, human DSB formation and recombination appear to be dominated by hotspots accounting for only a small proportion of the total genome and located most frequently in IGRs of the genome [40,41]. Human hotspots are located 50–100