Figure 3.

Dmc1 ChIP. The average hotspot:coldspot ratios corrected to input DNA are shown for each strain (n = 3). (A) Preferential association of Dmc1 at the HIS4::LEU2 hotspot depends on SPO11. Dmc1 enrichment was not detected in control experiments without cross-linking and without antibody (data not shown). Hotspot and coldspot signal is not detectable in strains lacking DMC1. Dmc1 is not enriched at hotspots in spo11Δ tid1Δ mutants, but twofold more DNA is immunoprecipitated at both the hotspot and the coldspot after 4 h in sporulation medium vs. the spo11Δ single mutant. (B) Dmc1 does not preferentially associate with DSBs at HIS4 ::LEU2 in a tid1Δ rad54Δ double mutant. “WT 20%” represents a mixture of cells consisting of 20% wild-type cells from 4 h in meiosis with 80% wild-type cells from a mitotic culture. This mixture was designed to mimic the low efficiency of meiotic induction in the tid1Δ rad54Δ mutant in order to demonstrate that the ChIP assay is capable of detecting preferential hotspot association, even when meiotic induction is inefficient. (C) Dmc1 Western blot showing accuracy of mixing in the “WT 20%” sample compared with tid1Δ rad54Δ.