may be a universal aspect of meiotic recombination. A hotspot created by the ade6-M26 mutation in S. pombe has been especially informative. The single bp mutation M26 creates a binding site for the transcription factor Atf1-Pcr1, which is essential for hotspot activity and DSB formation at M26 [6–9]. Although a few meiotic hotspots in S. pombe are binding sites for this transcription factor [9,24], most hotspots are not, since only a few DSBs depend on Atf1-Pcr1. Similarly, in S. cerevisiae a few hotspots depend on the Bas1 transcription factor . The determinants for the majority of the hotspots have been unclear.
To determine the global distribution of DSBs in S. pombe, we have used two methods—an extensive analysis of DSBs by Southern blot hybridizations and a genome-wide microarray analysis of Rec12-DNA linkages. We find that these two methods agree remarkably well and show, surprisingly, that the small class of exceptionally large IGRs is highly predictive and contains the majority of meiotic DSB hotspots.
Evidence for Meiosis-Specific Covalent Linkage of Rec12 to Hotspot DNA
Previous evidence suggested that Rec12, like S. cerevisiae Spo11, becomes covalently linked to DNA during meiosis. Rec12 Tyr98 corresponds to a Tyr residue present in all analyzed Spo11 proteins , and the alteration Tyr98 → Phe98 abolishes meiotic recombination . The corresponding Tyr135 of S. cerevisiae Spo11 is essential for recombination and DSB formation and is thought to be the active site residue linked to DNA . To confirm Rec12-DNA covalent linkage, we analyzed, by locus-specific PCR, DNA in immunoprecipitates (IPs) of chromatin from rec12-FLAG rad50S cells without an exogenous crosslinking agent; the Rec12-FLAG protein is fully active in meiotic recombination (Table S1). We analyzed DNA at two known hotspots—the M26-related ade6–3049 mutational hyper-hotspot  and the natural mbs1 hotspot . We also analyzed the ura1 locus, at which DSBs are not detectable by Southern blot hybridization . At both hotspots, but not at ura1, strong PCR signals were observed at 4, 5, and 6 h after meiotic induction (Figure S1), in parallel with the accumulation of DSBs at these times in rad50S cells as measured by Southern blot hybridizations . This meiosis-specific signal was dependent on the FLAG antibody (unpublished data), the FLAG tag on Rec12, and intracellular Rec10 protein, which is also essential for recombination and DSB formation . Approximately 30 times more signal was observed with primers specific to ade6, which contains a DSB hotspot, than with primers specific to ura1, which lacks detectable DSBs. As expected, approximately equal yields of product using the whole cell extract (WCE) (nonimmunoprecipitated) DNA were obtained at the ade6–3049, mbs1, and ura1 sites. Since no crosslinking agent such as formaldehyde was used, we infer that the observed PCR signals using the IPs reflect covalent linkage or very tight binding of Rec12 specifically to DNA at meiotic hotspots.
Genome-Wide Analysis of Rec12-DNA Linkages
To extend this analysis to the entire genome, we analyzed DNA in IPs of chromatin prepared from cells harvested before and 5 h after meiotic induction, using tiling microarrays of oligonucleotides (60-mers; “probes”) spaced on average every 300 bp across the S. pombe genome . These arrays have been used to analyze the genome-wide distribution of heterochromatin proteins and histone modifications . We have analyzed three meiotic inductions, two with a haploid strain (Dataset S1) and one with a diploid (Dataset S2). These strains are pat1–114 (Ts) mutants, which initiate meiosis synchronously when the temperature is raised, even from the haploid state ; where tested, haploids and diploids give DSB patterns indistinguishable by Southern blot analysis .
Using the tiling microarrays, we compared the Rec12-DNA signals obtained using the IPs to those obtained using WCEs. The ratio of the IP:WCE signals at each probe reflects the enrichment of DNA linked to Rec12. This ratio was normalized using the median values of the IP and WCE signals for all