), we consider sites with ≥0.5% estimated DSBs, based on the relation in Figure 4, to be “prominent” DSB hotspots or Rec12 peaks and those with <0.5% to be “weak.” From the peak integrals we estimate that the prominent Rec12 peaks represent ∼90% of all detectable Rec12-DNA linkages (see below). In the Discussion, we describe additional differences between the prominent and weak hotspots, which suggest that they are distinct.
DSB Hotspots Preferentially Occur in Large IGRs
Young et al.  noted that the DSB hotspot mbs1 is located in an IGR that is especially large—7.2 kb. The mode of S. pombe IGRs is 0.4 kb, and the median is 0.7 kb . mbs2 is also in a large IGR—6.3 kb. We therefore examined the microarray data for the location of hotspots and found a strong correlation between DSB hotspots and large IGRs. (For this analysis we classed as intergenic the putative coding sequences annotated as “dubious” or “very hypothetical.”) For example, in the 150-kb interval that includes mbs1 and mbs2 there are no other prominent hotspots and no other IGRs larger than 1.9 kb (Figure 5B). A nearby 150-kb interval on the left arm of Chromosome I has four prominent hotspots, which fall in IGRs of size 5.2, 4.3, 4.1, and 2.3 kb (Figure 5A). In these 300 kb all IGRs >2.8 kb have prominent hotspots, and five out of six such hotspots occur in IGRs >4 kb. In the 1.8-Mb region depicted in Figure 3, 16 of the 25 prominent Rec12 peaks confirmed by Southern blot analysis fall in IGRs >2.8 kb, five fall in IGRs of 0.6–2.3 kb, and the remaining four fall in coding sequences.
Microarray data for haploid 0-h DNA (blue line), the mean of two 5-h DNA analyses (red line) processed as in Figure 1, and coding sequences (genes, black lines at top) from the annotated S. pombe genome (Build 1.1; http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=genomeprj&cmd=Retrieve&dopt=Overview&list_uids=9517) are shown for two 150-kb intervals of Chromosome I.
(A) An interval to the left of that containing mbs2–mbs1 (see Figure 1).
(B) The mbs2–mbs1 region.
Carets indicate weak (black) and prominent (red) Rec12 peaks identified by PeakFinder.
We extended this analysis genome-wide, except for the telomeres and centromeres, which are considered separately in the Discussion. Rec12-DNA linkage positions were identified as the maxima of Rec12 peaks using the PeakFinder program . Using the mean of the haploid 5-h data, this program identified 353 such positions genome-wide. The 149, 124, and 80 positions seen on Chromosomes I, II, and III respectively (Figure S7) are in good agreement with the relative sizes of the chromosomes (5.6, 4.5, and 2.5 Mb, not including the rDNA at the ends of Chromosome III). Of the 353 Rec12 peak positions from the haploid data, 340 (96%) were also identified in the diploid data. Using a cutoff of p < 10−8, a cutoff that excludes all but 18 (12%) of the false-positive peaks identified in the 0-h data, the ChIPOTle program  independently identified peak regions in the haploid data that included all of the positions identified by PeakFinder, plus an additional 16 (4%). We used the peak positions identified by PeakFinder from the haploid data for the analysis below.
As mentioned above, we estimated the relative frequency of Rec12-DNA linkage at each hotspot by integrating the IP:WCE ratios across each peak and classified the peaks as “prominent” (≥0.5% estimated DSBs) or “weak” (<0.5%) based on the relation in Figure 4. Among the 353 peaks identified by PeakFinder, 194 are prominent hotspots. Based on the sum of the integrals, these prominent peaks account for 88% of all detectable Rec12-DNA linkages (DSBs).
Our analysis shows that DSB hotspots are over-represented in large IGRs and under-represented in coding DNA. Figure 6A shows the proportion of all genomic DNA and the proportion of all prominent and weak Rec12 peaks found in coding sequences and in IGRs of different sizes. The distributions of all DNA and Rec12 peaks are clearly distinct (for prominent peaks p < 0.0001 and for weak peaks p < 0.005; two-tailed contingency chi-squared test). IGRs >2 k