Figure 1.
Rec12-DNA Linkages Occur at Strong Hotspots Separated by Cold Regions across NotI Fragment J (524-1025 kb from the Left End of Chromosome I)
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 [14]. 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.
Figure 2.
Rec12-DNA Linkages Strongly Correlate with DSBs Determined by Southern Blot Hybridization
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 [5].
(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 [9].
Figure 3.
Close Correlation between DSBs and Rec12-DNA Linkages across 1.8 Mb of Chromosome I
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).
Figure 4.
Hotspot Strengths Measured by Southern Blots of DSBs and by Microarray Data of Rec12-DNA Linkages Correlate Well
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.
Figure 5.
DSB Hotspots Occur in Large IGRs
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.
Figure 6.
Most DSB Hotspots Occur in Large IGRs
Frequency distributions of DNA (green bars), weak Rec12-DNA linkage sites (gray bars), and prominent Rec12-DNA linkage sites (red bars) are shown for the classes of DNA sequence indicated. Analysis was carried out using the whole genome excluding the centromeres and 50 kb at the end of each chromosomal contig (to exclude telomeric regions).
(A) Number distribution. Peaks of Rec12-DNA linkage (DSB hotspots) identified from the mean of the haploid 5-h data were classified according to their position in coding sequences (CDS) or in IGRs. A total of 62% of prominent Rec12 peaks occur in IGRs >2 kb.
(B) Probability of a prominent Rec12 peak occurring within an IGR increases with IGR size.
(C) Density of prominent Rec12 peaks (number of peaks per kb) increases with IGR size.
(D) DSB density (Rec12 peak integrals per kb) is greatest in large IGRs.