that might correlate with DSB hotspots and thus be important factors for their determination. The meiosis-specific cohesin subunit Rec8 is required for most DSB formation in S. pombe . Rec8 is thought to be part of the chromosomal core that forms the basis of the axial elements (linear elements in S. pombe) and from which chromatin loops emanate . Studies in S. cerevisiae have led to the conclusion that DSB hotspots preferentially occur in the loops, where Rec8 density is low . S. pombe Rec8 is distributed nonuniformly, at least over the portion of the genome surveyed (Chromosome II and part of Chromosome III) . We observed a weak negative correlation between Rec8 binding and Rec12-DNA linkage. For example, 83% of the 69 prominent Rec12 peaks on Chromosome II are centered on an 11-probe window whose Rec8-binding ratio is below the median for Chromosome II (ignoring the centromere), significantly different from the 50% expected (p < 0.001; contingency chi-squared test). Among the 55 weak Rec12 peaks, this bias falls to 64%, not significantly different from 50%. Conversely, the quarter of probes on Chromosome II with the highest Rec8 enrichment ratios contain only 9% of the prominent Rec12 peaks, less than the 25% expected, and the quarter with the lowest Rec8 enrichment ratios contain 55% of the prominent Rec12 peaks, more than the 25% expected. Therefore, in S. pombe as in S. cerevisiae, DSBs are associated with regions of low Rec8 localization, but neither distribution is very useful for predicting the other.
As noted above, Atf1-Pcr1 and perhaps other transcription factors are required for some DSB hotspots. We therefore looked for a correlation between DSB hotspot positions and the promoters of genes induced during meiosis. Among such genes , we found no significant enrichment of Rec12 peaks 5′ of the corresponding open reading frame. We then looked among all genes with Rec12-peaks in their presumptive promoters for enrichment of related functions, as indicated by related gene ontology (GO) terms. Using the GOstat 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 