shorter DNA fragments gave a slightly different picture. The positions of the peaks at two well-studied DSB hotspots, mbs1 on Chromosome I and ade6–3049 on Chromosome III, again were coincident, but the Southern blot analysis of shorter DNA fragments now provides higher resolution than the microarray data (Figure 2B and 2C), as expected from the length of DNA linked to Rec12 (see above).
Haploid microarray data as in Figure 1 are plotted with tracings of a Southern blot of DNA that was prepared during a meiotic induction at the indicated times, subjected to gel electrophoresis, blotted, probed, and analyzed by Phosphorimager.
(A) NotI-digested DNA probed from the left end of 501-kb fragment J .
(B) MluI-digested DNA probed from the right end of the 20.9-kb fragment with mbs1.
(C) AflII-digested DNA probed from the right end of the 6.6-kb fragment with ade6 .
We extended this dual analysis beyond the DSB hotspots on NotI fragment J noted by Young et al. . We first analyzed DSBs by Southern blot analysis of the NotI fragments adjacent to fragment J (Figure S6). This analysis encompassed ∼1.8 Mb of the left arm of Chromosome I, including NotI fragment J, and represented ∼15% of the S. pombe genome. We found 25 discrete DSB hotspots, or tight clusters of hotspots, scattered across the region. These hotspots were then mapped more precisely by analysis of smaller restriction fragments, ∼10–100 kb long, covering much but not all of the 1.8 Mb region. Every DSB hotspot was coincident with a hotspot of Rec12-DNA linkage independently obtained by microarray analysis (Figure 3). We did not survey at higher resolution all of the weak Rec12-DNA linkage peaks or stronger DSB sites whose diagnostic DNA fragments were poorly separated from those of nearby DSB sites. Except for these few Rec12-DNA linkage sites that have not been tested by Southern blot analysis, there is a strict correspondence of Rec12-DNA linkage and DSB hotspots.
Microarray data from 0 h (blue line) and 5 h (red line) analyses of haploids are presented as in 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