onal strong hotspots of Rec12-DNA linkage were observed at six other sites to the right of the mbs2–mbs1 pair. This pattern was consistent for all three inductions, and the hotspots were essentially absent in the uninduced (0-h) samples (Figure S5). Between the obvious hotspots in the 5-h DNA the signals varied in a small range around a ratio of 1, similar to the 0-h data and consistent with the expected background (non-Rec12 enriched) distribution . Thus, the microarray analysis reveals strong hotspots of Rec12-DNA linkage separated by large regions in which linkage is undetectable (see additional discussion below).
Haploid strain GP6013 bearing the rec12–201::6His-2FLAG allele was induced and analyzed for Rec12-DNA linkage by microarray analysis before (0 h; blue line) and 5 h (red line; mean of two experiments) after meiotic induction. Data are the median-normalized IP:WCE ratios for each oligonucleotide, spaced ∼300 bp apart . Probes with ratios >2 were replaced with values of 1 unless they occurred in contiguous groups of >2 spots (see text for justification); high values within contiguous groups (hotspot peaks) in the 5-h data were omitted if they were also spuriously high in the 0-h data. See Figure S2 for alternative data presentations, Figure S5 for repeat experiments, and Figure S7 for the entire genome. Carets indicate weak (black) and prominent (red) Rec12 peaks identified by PeakFinder. Left and right vertical axes are offset to allow separation of the 0- and 5-h datasets.
Rec12-DNA Linkage Analysis by Microarrays Closely Parallels Direct DSB Analysis by Southern Blot Hybridization
We directly compared the distribution of Rec12-DNA linkages to that of DSBs determined by Southern blot analysis. A close correlation of both positions and intensities was seen across NotI fragment J (Figure 2A). At this scale (50–500-kb DNA fragments), the microarray data provide higher resolution and a higher signal-to-noise ratio than Southern blot analysis. Comparison of the microarray data with direct DSB analysis of 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 