hizosaccharomyces pombe, we analyze an event, the formation of DNA double-strand breaks (DSBs), which is essential for meiotic recombination. We find that hotspots of DSB formation occur primarily in unusually large intergenic regions (IGRs) and are widely separated by apparently break-free regions. Our results show that a clearly identifiable subset of noncoding DNA has a special function that is critical for meiosis. These results contrast with those obtained in the distantly related budding yeast Saccharomyces cerevisiae.
Meiosis consists of two special nuclear divisions during which chromosome number is reduced from two in somatic cells to one in gametes. Uniquely, in the first meiotic division homologs, rather than sister chromatids, segregate from each other. In most organisms homolog segregation is aided by crossover recombination.
Meiotic recombination is initiated by DSBs in both budding and fission yeasts and perhaps in all species [1–3]. Repair of the DSBs by interaction with a homolog produces the crossovers (reciprocal recombination events) important for homolog segregation and for the generation of genetic diversity. DSBs are formed by a meiosis-specific protein Spo11 (Rec12 in S. pombe) through a topoisomerase-like mechanism, in which a tyrosine residue of the protein forms a phosphodiester covalent linkage with each 5′ end at the DSB [3,4]. DSBs can be detected by extraction of DNA from meiotic cells and analyzing it by Southern blot hybridizations, which reveal distinct hotspots of break formation. DSBs are transient in wild-type cells but accumulate to high levels in mutants, such as rad50S, in which the Spo11 or Rec12 protein is not removed from the ends of breaks [2,5]. Analysis of DSBs is thus more sensitive in rad50S cells.
DSB hotspots were first detected at recombination hotspots, sites at which gene conversion (nonreciprocal recombination) occurs at especially high frequency. Gene conversion hotspots have been identified in the wild-type chromosomes of many species, including humans, and 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 P