 of the ∼41,000 probes . Because the chromatin DNA was sheared to ∼0.5–1 kb before immunoprecipitation, and because Spo11 (and likely Rec12) becomes linked to both 5′ ends at a DSB , we expect genuine Rec12-DNA signals to hybridize to at least three adjacent probes on the array. For our subsequent analysis we thus excluded isolated single or double probes (“spikes”) with high values (IP:WCE ratios >2), as done similarly by Cam et al. . High points in the induced (5 h) data corresponding to spuriously high uninduced (0 h) points were also removed. The effect of this filtering and its comparison with other noise-reducing methods are shown in Figure S2. High Rec12 enrichment ratios in the 0-h data are restricted to isolated spikes, whereas in the 5-h data high enrichment ratios are also seen in contiguous groups of high ratio probes, indicative of genuine Rec12 enrichment (Figure S10). Filtering removed the spikes (Figure S11). As expected , the filtered data from the 0-h samples were approximately normally distributed, whereas the data from the 5-h samples were approximately normally distributed but with an additional, substantial non-normal distribution of high values (i.e., sites of Rec12-DNA linkage) (Figures S3 and S4). Greater variance in the diploid 0-h background distribution meant that filtering of spikes was somewhat less effective in that dataset than in the others.
We first focused our attention on a 0.5-Mb region near the left end of Chromosome I, defined by NotI restriction fragment J, which we have previously analyzed extensively for meiotic recombination and DSBs . Our microarray analysis reveals a consistent distribution of Rec12-DNA linkages that closely parallels the distribution of DSBs determined by Southern blot hybridization (see next section). Strong Rec12-DNA signals (IP:WCE ratios >2 and up to ∼40) were observed at ∼20 consecutive probes at the position of the previously mapped mbs1 and mbs2 DSB hotspots of NotI fragment J in each of the three inductions (5-h data; Figures 1 and S5). Additional 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 