Acetylated lysine 56 on histone H3 drives chromatin assembly after repair and signals for the completion of repair

Abstract

DNA damage causes checkpoint activation leading to cell cycle arrest and repair, during which the chromatin structure is disrupted. The mechanisms whereby chromatin structure and cell cycle progression are restored after DNA repair are largely unknown. We show that chromatin reassembly following double-strand break (DSB) repair requires the histone chaperone Asf1 and that absence of Asf1 causes cell death, as cells are unable to recover from the DNA damage checkpoint. We find that Asf1 contributes toward chromatin assembly after DSB repair by promoting acetylation of free histone H3 on lysine 56 (K56) via the histone acetyl transferase Rtt109. Mimicking acetylation of K56 bypasses the requirement for Asf1 for chromatin reassembly and checkpoint recovery, whereas mutations that prevent K56 acetylation block chromatin reassembly after repair. These results indicate that restoration of the chromatin following DSB repair is driven by acetylated H3 K56 and that this is a signal for the completion of repair.

Figure 1. DNA resection drives chromatin disassembly around a DSB

A. Schematic of mating type loci in strain JKM179 and positions of primers used for DNA repair and ChIP analyses below. The HMR and HML donor loci are deleted in strain JKM179. B. PCR assay of DNA cutting and failure to repair following induction of the HO endonuclease by addition of galactose at Time = 0hr. Below is quantitation of three independent experiments, after normalization to the control. C. Analysis of DNA levels and H3 levels flanking the double-strand break. The left panel shows input DNA used for the ChIP analysis 0.6kb from the HO site normalized to a distal SMC2 site. The middle panel shows the amount of DNA 0.6kb from the HO site from the H3 ChIP analysis (“H3 IP”), normalized to a distal SMC2 site. The right panel shows the normalization of the H3 IP to input signals. D. As for C, but in the wild type strain carrying H3-FLAG (YTT035). In addition, the right panel shows quantitation of cutting and repair, determined as in B. E. As for D, but in the arp8Δ strain carrying H3-FLAG (BAT058). F.. As for D, but in the mre11Δ strain carrying H3-FLAG (BAT061). G. As for D, but in the asf1Δ strain carrying H3-FLAG (BAT062). H. The left panel shows a plot of all the input DNAs from panels D–G, while the right panel shows a plot of all the DNA from the H3 ChIPs from panels D–G. Note that the wild type H3 ChIP data was normalized to 1 at time 0.5 hrs rather than time 0 hrs to better enable comparison between the strains.

Figure 2. Asf1-dependent reassembly of chromatin following DSB repair

A. Schematic of mating type loci, showing positions of PCR primers generating MATa and MATα products for assaying DNA cutting and repair, and positions of primer pairs used for histone ChIP. B. Gel and quantitation of DNA cutting and repair in a wild type strain (BAT009), as described in Fig. 1. Galactose was added at time 0 to induce HO endonuclease and glucose added at 2 hours to allow repair using the donor sequences at HMR and HML. C. Chromatin disassembly and reassembly during DNA repair in wild type yeast (BAT009) at 0.6kb from the HO site. The input DNA is shown in the left panel. Quantitation of the ChIP (“H3 IP”) analysis of histone H3 is shown in the right panel, normalized as described in Fig. 1. D. As for C but at 2.0 kb from the HO site. E. As for C but in a strain deleted for RAD52 (JLY075). The right panel shows the HO cutting and repair analysis from the same time course. F. As for E but in a strain deleted for ASF1 (BAT063).

Figure 3. Delayed cell cycle re-entry in asf1 mutants following DSB repair

A. Asynchronous cultures of WT (JKT010) and asf1Δ (JKT018) yeast were exposed to MMS for 2 hours, which causes a G2/M accumulation. Flow cytometry analysis of DNA content was used to follow the cell cycle distribution after washing out the MMS. B. Schematic for system used to measure SSA. Repair of the HO lesion at the HO-cs site requires 5 kb of resection back to the uncleavable HOcs-inc site. The position of PCR primers and products used to measure repair in panel C are shown. C. The Rad53 kinase remains activated in asf1 mutants. The DSB was induced at time 0 by addition of galactose to wild type (YMV045), rad52Δ (YMV046) and asf1Δ strains (JKT200). The top panels show analysis of the repair of the HO lesion (note that the cut DNA gives no product). The lower panels show western analyses of Rad53 at the same time points. “Xreaction” refers to a cross-reacting protein that serves as a normalization control for loading. D. Chromatin disassembly and reassembly analysis using the identical strains and time course shown in C. The top panels show the input, and the lower panels show the ChIP analysis of H3, normalized as in Fig. 1.

Figure 4. asf1 mutants have a defect in recovery from the DNA damage checkpoint

A. 10-fold serial dilution analysis of the indicated strains, showing that asf1 mutants are sensitive to a galactose-induced unique HO endonuclease cut. Strains WT (YMV002), asf1Δ (JCY001), rad52Δ (YMV037), srs2Δ (YMV057), and kuΔ (YMV2-1) required 30kb of resection during repair by SSA, while strains WT (YMV045), asf1Δ (JKT200), and rad52Δ (YMV046) required 5kb of resection during repair by SSA. B. Quantitation of colony size formation from single cells following the indicated length of times of growth on galactose-containing plates, in WT (YMV002), asf1Δ (JCY001), rad52Δ (YMV037), and srs2Δ (YMV057) strains.

Figure 5. Chromatin reassembly is not required for removal of Mec1-Ddc2 or phosphorylated H2A after DNA repair, but is required for checkpoint adaptation

A. Removal of Ddc2 from the site of DNA repair does not require chromatin assembly. The HO lesion was induced in strains WT (JFY016) and asf1Δ (JFY017) at time 0 by addition of galactose. The level of Ddc2 flanking the HO lesion during SSA repair was measured by ChIP analysis. B. Loss of phosphorylated H2A from chromatin does not require Asf1. The HO lesion was induced in strains WT (YMV045), asf1Δ (JKT200), and rad52Δ (YMV046) at time 0 by addition of galactose. The level of H2A phosphorylated on serine 129 flanking the HO site in strains undergoing HO repair by SSA was measured by ChIP analysis. C. Asf1 contributes to checkpoint adaptation. Colony formation was assessed at the indicated times after placing single unbudded cells onto galactose plates to induce the unrepairable DSB in isogenic WT, kuΔ and asf1Δ strains derived from JKM179.

Figure 6. Acetylated H3 K56 is required for chromatin assembly and DNA damage checkpoint recovery after DNA repair

A. Mimicking H3 K56 acetylation can bypass the requirement for Asf1 for resistance to double-strand DNA damage. 10-fold serial dilution analysis of the indicated isogenic strains. B. Rtt109 is required for recovery from the DNA damage checkpoint. The same analysis presented in Fig. 3 was performed on WT (YMV045) and rtt109Δ (JFY013) strains undergoing SSA with 5kb of resection. C. Rtt109 is required for viability after repair of the HO site. 10 fold serial dilution analysis of WT (YMV045), asf1Δ (JKT200), rad52Δ (YMV046) and rtt109Δ (JFY013) strains was performed as described in Fig. 4A. D. Rtt109 is required for recovery from the DNA damage checkpoint. Colony formation analysis of WT (YMV045), asf1Δ (JKT200) and rtt109Δ (JFY013) strains as described in Fig. 4B. E. Rtt109 is required for chromatin reassembly after DNA repair. Analysis of cutting / repair and chromatin assembly and disassembly was performed on strain rtt109Δ (JFY013) as described in Fig. 2. F. A mimic of permanent H3 K56 acetylation bypasses the requirement for Asf1 for chromatin reassembly after DNA repair. Analysis of cutting / repair and chromatin assembly and disassembly was performed on strain asf1ΔK56Q as described in Fig. 2.

Figure 7. Model for the role of chromatin in deactivation of the DNA damage checkpoint