stat Web server (http://gostat.wehi.edu.au)  with Benjamini correction for multiple hypothesis testing, we found a significant enrichment for terms, such as GO:0051704 (Interaction between organisms), GO:0000746 and GO:0019953 (Conjugation and Sexual reproduction, subsets of GO:0051704), and GO:0003700 (Transcription factor activity), listed in Table S2. The causal basis of these correlations is not obvious. However, the enrichment of Rec12 peaks in the promoter regions of genes associated with conditions and processes related to meiosis and meiotic induction suggests that meiosis-specific chromatin remodeling may occur at some promoters, allowing access or activation of Rec12 for DSB formation. A particular group of transcription factors and transcription factor-binding sequences may be involved. As noted above, this is the case for the M26 hotspot and Atf1-Pcr1, factors involved in the meiosis-specific remodeling of chromatin and Rec12-dependent DSB formation [31–33].
In S. pombe mitotic replication origins are spaced about 25–30 kb apart, and, where tested, the same origins are used during both mitotic growth and meiosis . We found, however, no obvious relationship between origins of replication and meiotic DSB hotspots. For example, 14% of all replication origins correspond to haploid 5-h probe positions with a Rec12 IP:WCE ratio >2 (i.e., all regions of Rec12-DNA linkages), not significantly different from the number expected based on the proportion (11%) of all probes with a Rec12 enrichment value >2 (p > 0.05; contingency chi-squared test). Therefore, the frequency of origins of replication is neither enriched nor depleted significantly in regions of Rec12-DNA linkage.
We looked for an unusual nucleotide sequence composition surrounding DSB hotspots and found only a weak correlation with higher than average guanine-cytosine (GC) content. For example, 5-kb windows centered on the 194 prominent Rec12 peak positions have a slightly enriched GC content compared to 5-kb windows, moved in 100-bp steps, covering the whole genome (median of 36.6% versus 36.0%; p < 0.0001 by two-tailed t-test). However, base composition is a very poor predictor of prominent Rec12 peak position, as 37% of prominent Rec12 peaks are centered on windows with GC content below the global median and only 0.2% of windows with GC content above the global median are centered on a prominent Rec12 peak. No significant GC content enrichment was seen at weak Rec12 peak positions. Sliding windows of 0.5 kb, 1 kb, or 10 kb showed even less difference between peak-centered and whole-genome window compositions (unpublished data). In S. cerevisiae, DSB hotspots are also weakly correlated with higher than average GC content when a 5-kb sliding window is used .
Comparison of S. pombe, S. cerevisiae, and Human DSB Hotspot Distributions
As in S. pombe, DSBs have been analyzed in S. cerevisiae by both direct Southern blot analysis and microarray analysis of DNA covalently linked to Spo11, the Rec12 homolog [e.g.,20,21,35]. These analyses show that DSB hotspots in S. cerevisiae are in general slightly less prominent and often more closely spaced than those in S. pombe. For example, on the most thoroughly analyzed S. cerevisiae Chromosome III, the maximal fraction of DNA broken at a hotspot is 9% , and the maximal enrichment of Spo11-DNA linkage is 8-fold  or 15-fold . In many regions of the genome, most of the open reading frames have a detectable DSB at their 5′ end.
In S. pombe many DSB hotspots have 10%–15% of the DNA broken (Figures 4 and S6) , and Rec12-enrichment factors (IP:WCE ratio) are often >25 and occasionally up to 50 (Figures 3 and S7). Between the prominent Rec12 peaks, there are frequent intervals of 50–100 kb with few if any DSBs detectable by either Southern blot or microarray analysis (Figures 3 and S7) . We have not noted continuous stretches of genes with DSB hotspots at their 5′ ends. Thus, DSB hotspots (or small groups of hotspots) are usually isolated from others in S. pombe. In spite of hotspots being far apart, crossovers in S. pombe are 