Analysis of Protein–DNA Interactions During Meiosis by Quantitative Chromatin Immunoprecipitation (qChIP)
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During meiotic prophase a number of important events require recombination between maternal and paternal chromosomes, which is initiated through the introduction of DNA double-strand breaks (DSBs). The majority of DSBs, which mostly occur at so-called hotspots, have been located between cohesin binding sites. qChIP (chromatin immunoprecipitation quantified by real-time PCR) is a sensitive, accurate, and cost-efficient alternative to ChIP-on-Chip for the analysis of noncovalent protein–DNA interactions at defined binding sites in vivo. Here we use qChIP to study Mre11 binding to three chromosomal loci during meiosis. We show that Mre11 interacts with a known hotspot region (YCR048) in the R-band of chromosome III, but not with a cold region in the G-band (YCR011). Interestingly Mre11 binds to a cohesin binding site (YCR067 ), 20 kb distal to YCR048, with similar intensity as to the hotspot, despite the absence of DSBs in this region.
Key wordsChIP qChIP real-time PCR qPCR DNA binding Mre11 meiotic recombination double-strand break repair
We thank Marie Therese Hauser, Kim Nasmyth, and Katsuhiko Shirahige for helping us to set up, or improve parts of the technique and former lab member Silvia Prieler for tagging Mre11. This work was supported by FWF grant P18847 to F.K.