Chromatin remodelling at a DNA double-strand break site in Saccharomyces cerevisiae

Abstract

The repair of DNA double-strand breaks (DSBs) is crucial for maintaining genome stability. Eukaryotic cells repair DSBs by both non-homologous end joining and homologous recombination. How chromatin structure is altered in response to DSBs and how such alterations influence DSB repair processes are important issues. In vertebrates, phosphorylation of the histone variant H2A.X occurs rapidly after DSB formation, spreads over megabase chromatin domains, and is required for stable accumulation of repair proteins at damage foci. In Saccharomyces cerevisiae, phosphorylation of the two principal H2A species is also signalled by DSB formation, which spreads approximately 40 kb in either direction from the DSB. Here we show that near a DSB phosphorylation of H2A is followed by loss of histones H2B and H3 and increased sensitivity of chromatin to digestion by micrococcal nuclease; however, phosphorylation of H2A and nucleosome loss occur independently. The DNA damage sensor MRX is required for histone loss, which also depends on INO80, a nucleosome remodelling complex. The repair protein Rad51 (ref. 6) shows delayed recruitment to DSBs in the absence of histone loss, suggesting that MRX-dependent nucleosome remodelling regulates the accessibility of factors directly involved in DNA repair by homologous recombination. Thus, MRX may regulate two pathways of chromatin changes: nucleosome displacement for efficient recruitment of homologous recombination proteins; and phosphorylation of H2A, which modulates checkpoint responses to DNA damage.

Figure 1

Chromatin changes at the MATαDSB.(a) A DSB was induced at MATα in Flag-H2B or Flag-H3 expressing strains, and ChIP was performed with γ-H2A or anti-Flag antibodies. DNA was analyzed by real-time PCR using primers corresponding to sequences on the left (“−”) or right (“+”) side of the DSB (“0”), and results were normalized to the 0′ IP/Input DNA. Data in graphs are means +/− s.e.m. (b) Nuclei were prepared after DSB induction, and chromatin was digested with MNase and subjected to Southern blot analysis using a MATα DNA probe. M=1 Kb DNA ladder.

Figure 2

MRX plays a role in histone loss at the MATαDSB.(a) A DSB was induced at MATα, and ChIP was performed with anti-Flag antibodies in an mre11::Kan-MX strain expressing Flag-H2B and an hta1/hta2-S129* strain expressing Flag-H3. DNA was analyzed by realtime PCR on both the left and right sides of the break site (“0”), and data are means +/− s.e.m.. (b) MNase analysis was performed on nuclei isolated from the mre11::Kan-MX strain by Southern blot analysis as described in Figure 1c.

Figure 3

The INO80 complex is required for histone eviction at the MATα DSB.(a) A DSB was induced at MATα in an arp8::Kan-MX mutant expressing Flag-H3, and ChIP was performed with antibodies against the Flag epitope. Precipitated DNA was quantitated as described in Figure 1a. (b) ChIP was performed with anti-Myc antibodies in a wild type strain that contained Ino80-Myc before (−Gal) and after (+Gal) DSB induction. Ino80-Myc association was normalized to histone H3 occupancy. (c) MNase digestion was performed on nuclei isolated from an arp8::Kan-MX mutant as described in Figure 1c. Data in graphs are means +/− s.e.m.

Figure 4

MRX and INO80 are required for recruitment of Rad51 to the MATα DSB.(a) ChIP was performed in wild type, arp8Δ, or mre11Δ with RPA (left) or Rad51 (right) antibodies after DSB induction at MATα. DNA on the right side of the DSB was analyzed by real-time PCR, and data are means +/− s.e.m.. (b) MRX controls chromatin remodeling at DSBs. MRX is recruited to DSBs, regulating DNA end processing and Tel1-depenedent γ-H2A formation. MRX regulates nucleosome-remodelingthrough INO80, leading to nucleosome eviction and efficient recruitment of HR proteins. γ-H2A is independent of nucleosome displacement and controls accumulation of checkpoint proteins, as well as cohesin, at DSBs.