nearly uniformly distributed . Crossing-over far from detectable DSBs may involve migration of DNA intermediates far from a DSB, or they may be initiated by DNA lesions not detectable by current technology [12,36].
In these two yeasts meiotic DSBs are distributed differently in the centromeric and telomeric regions than in other regions of the genome. In S. cerevisiae centromeres are small (∼0.1 kb), and DSBs are infrequent to one or both sides of many of the centromeres [10,20,21,35]. In S. pombe centromeres are large (35–100 kb), and DSBs are undetectable within them by microarray analysis (Figure S7) or by Southern blot analysis (C. Ellermeier and G. Smith, unpublished data). In the adjacent intervals, however, DSBs appear as frequent as in other regions of the genome. In both yeasts ChIP-on-chip analysis suggests that Rec12 (Spo11)-DNA linkages are under-represented in the terminal 40 kb by ∼20% (unpublished data) [10,20,21]. In S. pombe, however, there is a prominent DSB ∼18 kb from the left end of Chromosome I, including ∼10 kb of unsequenced DNA (Figure 3), although prominent Rec12 peaks occur only 70–150 kb from the other three telomeres. DSB-free intervals of similar size occur throughout the chromosomes. Thus, with respect to meiotic DSBs S. pombe telomeres do not appear substantially different from other regions of the genome.
Meiotic DSBs have not been directly detected in human cells, but hotspots of recombination, presumably reflecting hotspots of DSB formation, have been detected using both linkage disequilibrium analysis and sperm typing. These methods have detected small regions of DNA, often only 1–2 kb wide by sperm typing, where meiotic recombination rates are greatly elevated compared to neighboring intervals or the genome average [37–40]. Like S. pombe and S. cerevisiae, human DSB formation and recombination appear to be dominated by hotspots accounting for only a small proportion of the total genome and located most frequently in IGRs of the genome [40,41]. Human hotspots are located 50–100 kb apart [39,42], similar to the distribution of hotspots in S. pombe (Figure 3). The strongest human hotspot has a crossover frequency of ∼1% , lower than that at mbs1 (5%)  and presumably than at several even stronger DSB hotspots in S. pombe (Figures 3 and S7). Human DSB hotspots are expected to be weaker than those of S. pombe, given the similar hotspot spacing and map sizes of the two organisms but greatly larger physical genome size of humans. Like S. pombe and S. cerevisiae, human hotpots are positively, but only weakly, correlated with GC content; this correlation has low predictive power . Therefore, several characteristics of meiotic DSB hotspots are common to S. pombe, S. cerevisiae, and humans, but there are also distinct differences in any pair-wise comparison of the organisms.
Further comparisons of meiotic DSB patterns in these yeasts and additional species will help understand how this process, crucial to sexual reproduction, is controlled. The analysis reported here provides a foundation for identifying the genomic features that regulate meiotic DSB formation, a problem so far unsolved for any organism.
Materials and Methods
S. pombe strains.
The genotypes of strains and the sources of published alleles are in Table S3. The rec12–201::6His-2FLAG allele, called rec12-FLAG in the text, was constructed as plasmid pJF32 (Text S1). The 1.6-kb BamHI fragment of pJF32 was used to transform strain GP5622 (rec12–171::ura4+) to fluoro-orotic acid-resistance to generate strain GP5960; the chromosomal allele was confirmed by automated nucleotide sequence analysis. The rec12–202::6His-2HA allele, called rec12-HA in the text, was similarly constructed as plasmid pJF41 and transferred to the chromosome to generate strain GP5961. Strains with these alleles are recombination proficient (Table S1). Other strains were constructed by standard meiotic crosses.
Preparation of meiotic chromatin and Rec12-FLAG covalently linked to DNA and its analysis with microarrays.
Cells were grown and induced for meiosis as descri