Figure 1. Numbered black diamonds indicate discernable DSB hotspots seen on Southern blots of DNA digested with a variety of enzymes that produce 10–100-kb fragments (see examples in Figures 2 and S6 for the Southern blots analyzed). Intensity and position data for mbs1 and mbs2 (numbers 7 and 8) are from [5,12]. Carets indicate weak (black) and prominent (red) Rec12 peaks identified by PeakFinder. The peak at 1,776 kb is at the rec12 gene and likely results from the rec12-FLAG allele used for microarray analysis; no DSB hotspot was evident by Southern blot analysis of rec12+ strains (Figure S7).
Signal Strengths by the Two Measures Are Similar
We noted above that the relative signal strengths of the peaks on the 0.5 Mb NotI fragment J were similar by Rec12-DNA and direct DSB analyses (Figure 2A). We extended this comparison for the 25 peaks seen by both measures in the 1.8 Mb region shown in Figure 3. We observed a strong trend between the strength of the DSB signals (percent of DNA broken at the site) and the strength of the microarray signals (IP:WCE ratios integrated over the peak) (Figure 4). The Pearson correlation coefficient r was 0.89 (p < 0.001 by t-test; r2 = 0.79); the Spearman rank correlation coefficient ρ was 0.90 (p < 0.001 by t-test).
The percent of total DNA broken at each of the 25 hotspots indicated by numbered black diamonds in Figure 3 is plotted versus the integral of the IP:WCE ratios for the corresponding peaks in the microarray data (mean of haploid and diploid experiments). The regression line is y = 8.35x + 10.4.
The data in Figures 2–4 show that the microarray data faithfully reflect direct assay of DSBs by Southern blot analysis, with respect to both the position of the DSBs and the amount of DNA breakage (strength of the hotspot). This outcome allows us to use the microarray data as a reliable substitute assay for DSBs across the entire genome. Hereafter, we use “DSB hotspots” and “Rec12 peaks” interchangeably. Because the limit of detection on our Southern blots is ∼0.5% (Figure S6), 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 