 relative to RAD50 . In S. pombe the number, position, and relative intensities of DSB hotspots are not significantly different in rad50S and rad50+ strains (Figure S9). This difference in the two yeasts may reflect Rad50 being required for DSB formation in S. cerevisiae but not in S. pombe [2,23]. Furthermore, the pat1–114 mutation, which allows a more nearly synchronous induction of meiosis than that in pat1+ strains, has little if any effect on the DSB pattern (Figure S9). Thus, the analyses presented here appear to reflect the DSB pattern in wild type.
Young et al.  analyzed extensively the 0.5-Mb NotI fragment J represented in Figure 1 and noted a noncongruence between the distribution of DSBs and crossovers. Our microarray data confirm this noncongruence, which remains an unsolved paradox.
The only known determinant of the position of meiotic DSB hotspots in S. pombe is the transcription factor Atf1-Pcr1 and its DNA binding site [24 and references therein]. Of the 15 genomic sequences closest to the consensus sequence for binding of Atf1-Pcr1 to naked DNA, 11 are DSB hotspots demonstrable by Southern blot hybridization , and we identified ten of these by microarray analysis (Figure S7) (unpublished data). We note, however, that the consensus sequence is not sufficient for DSB formation: four loci with this sequence do not have DSBs detectable by either method, and most ade6-M26 transplaced fragments with an Atf1-Pcr1 binding site are not recombination hotspots [25,26]. Nor is Atf-Pcr1 binding required for the DSB formation at most hotspots . Nevertheless, the ability to successfully predict DSB hotspots from the Atf1-Pcr1 consensus sequence encourages the search for other sequences or genomic features that determine hotspots.
Meiotic DSBs Are Concentrated in Large IGRs
The most prominent feature associated with DSB hotspots that we have noted is exceptionally large IGRs (Figures 5 and 6). Approximately 50% of the IGRs >3 kb in length have Rec12 peaks (Figure 6A), which collectively account for approximately 50% of all detectable Rec12-DNA linkages (DSBs). Thus, this genomic feature is highly predictive. We presume that one or more nucleotide sequences, currently unknown but restricted to the IGRs with DSBs, are essential for DSB formation. These sequences might be collections of transcription factor binding sites, exemplified by M26, the binding site for Atf1-Pcr1. The S. cerevisiae transcription factor Bas1 and its binding site also account for a small fraction of the meiotic DSB hotspots observed in that distantly related yeast . We think it likely that transcription factors other than Atf1-Pcr1 also influence DSB formation in S. pombe. Transcription factor binding sites are concentrated in IGRs and collectively may account for a significant fraction of meiotic DSBs. Other sequences, such as those affecting nucleosome positioning, may determine additional DSB hotspots. The data reported here provide a framework for identifying such determinants.
Ludin and colleagues also noted an enrichment of Rec12 in IGRs in their genome-wide analysis of Rec12 crosslinked with formaldehyde to meiotic DNA (K. Ludin, J. Mata, J. Bahler, and J. Kohli, personal communication). In their analysis Rec12 was more uniformly distributed across the genome than in our analysis, perhaps a reflection of only a fraction of chromatin-bound Rec12 being active in making DSBs and becoming crosslinked without an exogenous agent.
We observed that DSB hotspots are over-represented in IGRs whose flanking genes are divergently transcribed, compared to their frequency in all IGRs (p < 0.001; contingency chi-squared test). However, the orientation of transcription of adjacent genes is itself correlated with the size of the IGR , and no enrichment for DSB hotspots was seen in divergent IGRs when the size of the IGR was controlled for (unpublished data). We know of no clear evidence that transcription per se influences meiotic DSB formation (e.g., ).
Relation of Meiotic DSBs and Other Genomic Features
We searched for other features of the S. pombe genome 