b comprise only 13% of the genome, but they contain 62% of all prominent Rec12 peaks and 37% of the weak Rec12 peaks. Similarly, IGRs >3 kb comprise only 8% of the genome but contain 47% of the prominent Rec12 peaks and 19% of the weak Rec12 peaks. Thus, prominent peaks are more strongly enriched in large IGRs than are weak peaks. In fact, among prominent peaks, the probability of a peak lying in an IGR >3 kb rises with peak integral so that the very strongest peaks are even more highly associated with large IGRs (Figure S8). The Rec12 peak integrals, a measure of Rec12-DNA linkages and hence DSBs, are similarly skewed toward large IGRs: 65% of all Rec12-DNA linkages are in IGRs >2 kb, and 52% are in IGRs >3 kb. In addition, fewer prominent hotspots than weak hotspots (19% versus 38%) occur in coding DNA, which accounts for 61% of the genome.
Frequency distributions of DNA (green bars), weak Rec12-DNA linkage sites (gray bars), and prominent Rec12-DNA linkage sites (red bars) are shown for the classes of DNA sequence indicated. Analysis was carried out using the whole genome excluding the centromeres and 50 kb at the end of each chromosomal contig (to exclude telomeric regions).
(A) Number distribution. Peaks of Rec12-DNA linkage (DSB hotspots) identified from the mean of the haploid 5-h data were classified according to their position in coding sequences (CDS) or in IGRs. A total of 62% of prominent Rec12 peaks occur in IGRs >2 kb.
(B) Probability of a prominent Rec12 peak occurring within an IGR increases with IGR size.
(C) Density of prominent Rec12 peaks (number of peaks per kb) increases with IGR size.
(D) DSB density (Rec12 peak integrals per kb) is greatest in large IGRs.
Prominent DSB hotspots, as indicated by sites of Rec12-DNA linkage, are therefore particularly strongly associated with large IGRs. In addition, large IGRs are highly predictive of DSB hotspots, since 44% of the IGRs >3 kb long have prominent Rec12 peaks (Figure 6B) and an additional 12% have weak Rec12 peaks. Furthermore, prominent Rec12 peaks are about 20 times more dense (peaks per kb) in IGRs >3 kb than in coding sequences or short IGRs (Figure 6C). The density of DSBs (peak integrals per kb) is also 20–40 times greater in IGRs >3 kb than in coding sequences or in short IGRs (Figure 6D). We discuss below the significance of these observations.
A current critical question is which genomic features and underlying nucleotide sequences distinguish meiotic DSB hotspots from other portions of the genome. Our global microarray analysis permits a genome-wide analysis of meiotic DSBs in fission yeast. The distribution of meiotic DNA covalently linked to Rec12 is nearly identical to the distribution of meiotic DSBs determined directly by Southern blot hybridizations. This correspondence holds both for the presence of DSB hotspots (Figures 2 and 3) and for their relative strengths (Figure 4). We can thus use the more easily determined Rec12-DNA linkage data from microarray analysis as a valid proxy for DSBs genome-wide. Because the limit of detection on our Southern blots is ∼0.5% (Figure S6) , we concentrate on prominent Rec12 peaks, those whose integrals indicate a DSB frequency ≥0.5% (Figure 4). Our analysis of the 194 prominent hotspots that we identified in the 12.6-Mb S. pombe genome shows that prominent DSBs are detectable at hotspots spaced on average ∼65 kb apart (Figure S7). Between these hotspots there is very little, if any, Rec12-DNA linkage detectable (Figures 1–3 and 5) or DSBs detectable by Southern blot analysis (Figure 2A) (unpublished data) . We estimate from our microarray data that DSBs collectively occur in ∼700 kb of DNA, or ∼6% of the genome. Below, we discuss current knowledge of what is special about this small portion of the genome.
Most analyses of meiotic DSBs in S. pombe and S. cerevisiae have used the rad50S (K81I) mutation to allow accumulation of DSBs and thus a higher DSB level at most hotspots [20,21]. In one analysis using both RAD50 and rad50S S. cerevisiae, DSBs were reduced in some but not other intervals in rad50S