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Meiotic recombination is initiated by DNA double-strand breaks (DSBs) that are catalyzed by the type II topoisomerase-like Spo11 protein. Locally, at recombination hot spots, Spo11 introduces DSBs at multiple positions within ∼75 to 250 bp, corresponding to accessible regions of the chromatin. The molecular basis of this multiplicity of cleavage positions, observed in a population of meiotic cells, remains elusive. To address this issue, we have examined the properties of the Gal4BD-Spo11 fusion protein, which targets meiotic DSBs to regions with Gal4 binding sites (UAS). By single-nucleotide resolution mapping of targeted DSBs, we found that DSB formation was restricted to discrete sites approximately 20 nucleotides from the UAS, defining a “DSB targeting window.” Thus, the multiplicity of cleavage positions at natural Spo11 hot spots likely represents binding of Spo11 to different distinct sites within the accessible DNA region in each different meiotic cell. Further, we showed that mutations in the Spo11 moiety affected the DSB distribution in the DSB targeting window and that mutations in the DNA at the Spo11 cleavage site affected DSB position. These results demonstrate that Spo11 itself has sequence preference and contributes to the choice of DSB positions.

Locally, Meiotic Double-Strand Breaks Targeted by Gal4BD-Spo11 Occur at Discrete Sites with a Sequence Preference