 peaks are about 20 times more dense (peaks per kb) in IGRs >3 kb than in coding sequences or short IGRs (Figure 6C). The density of DSBs (peak integrals per kb) is also 20–40 times greater in IGRs >3 kb than in coding sequences or in short IGRs (Figure 6D). We discuss below the significance of these observations.
A current critical question is which genomic features and underlying nucleotide sequences distinguish meiotic DSB hotspots from other portions of the genome. Our global microarray analysis permits a genome-wide analysis of meiotic DSBs in fission yeast. The distribution of meiotic DNA covalently linked to Rec12 is nearly identical to the distribution of meiotic DSBs determined directly by Southern blot hybridizations. This correspondence holds both for the presence of DSB hotspots (Figures 2 and 3) and for their relative strengths (Figure 4). We can thus use the more easily determined Rec12-DNA linkage data from microarray analysis as a valid proxy for DSBs genome-wide. Because the limit of detection on our Southern blots is ∼0.5% (Figure S6) , we concentrate on prominent Rec12 peaks, those whose integrals indicate a DSB frequency ≥0.5% (Figure 4). Our analysis of the 194 prominent hotspots that we identified in the 12.6-Mb S. pombe genome shows that prominent DSBs are detectable at hotspots spaced on average ∼65 kb apart (Figure S7). Between these hotspots there is very little, if any, Rec12-DNA linkage detectable (Figures 1–3 and 5) or DSBs detectable by Southern blot analysis (Figure 2A) (unpublished data) . We estimate from our microarray data that DSBs collectively occur in ∼700 kb of DNA, or ∼6% of the genome. Below, we discuss current knowledge of what is special about this small portion of the genome.
Most analyses of meiotic DSBs in S. pombe and S. cerevisiae have used the rad50S (K81I) mutation to allow accumulation of DSBs and thus a higher DSB level at most hotspots [20,21]. In one analysis using both RAD50 and rad50S S. cerevisiae, DSBs were reduced in some but not other intervals in rad50S 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