Delineation of the role of chromatin assembly and the Rtt101^Mms1 E3 ubiquitin ligase in DNA damage checkpoint recovery in budding yeast

<div><p>The DNA damage checkpoint is activated in response to DNA double-strand breaks (DSBs). We had previously shown that chromatin assembly mediated by the histone chaperone Asf1 triggers inactivation of the DNA damage checkpoint in yeast after DSB repair, also called checkpoint recovery. Here we show that chromatin assembly factor 1 (CAF-1) also contributes to chromatin reassembly after DSB repair, explaining its role in checkpoint recovery. Towards understanding how chromatin assembly promotes checkpoint recovery, we find persistent presence of the damage sensors Ddc1 and Ddc2 after DSB repair in <i>asf1</i> mutants. The genes encoding the E3 ubiquitin ligase complex Rtt101^Mms1 are epistatic to <i>ASF1</i> for survival following induction of a DSB, and Rtt101^Mms1 are required for checkpoint recovery after DSB repair but not for chromatin assembly. By contrast, the Mms22 substrate adaptor that is degraded by Rtt101^Mms1 is required for DSB repair <i>per se</i>. Deletion of <i>MMS22</i> blocks loading of Rad51 at the DSB, while deletion of <i>ASF1</i> or <i>RTT101</i> leads to persistent Rad51 loading. We propose that checkpoint recovery is promoted by Rtt101^Mms1-mediated ubiquitylation of Mms22 in order to halt Mms22-dependent loading of Rad51 onto double-stranded DNA after DSB repair, in concert with the chromatin assembly-mediated displacement of Rad51 and checkpoint sensors from the site of repair.</p></div

Rtt101^Mms1 ubiquitin ligase functions in the same pathway as Asf1 in response to a DSB.

<p><b>A and B</b>. 10-fold serial dilution analysis of WT (YMV045), <i>asf1</i>Δ (JKT200), <i>rad52</i>Δ (YMV046), <i>rtt101</i>Δ (CCY019), <i>mms1</i>Δ (CCY018), <i>mms22</i>Δ (CCY024), <i>rtt107</i>Δ (CCY022), <i>asf1</i>Δ <i>rtt101</i>Δ (CCY026), and <i>asf1</i>Δ <i>mms1</i>Δ (CCY027) strains containing the SSA HO repair system was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180556#pone.0180556.g001" target="_blank">Fig 1B</a>. <b>C</b>. The HO endonuclease was induced by addition of galactose at 0hr in the WT (YMV045), <i>rtt101</i>Δ (CCY019), and <i>mms1</i>Δ (CCY018) strains. The top panels show an analysis of the cutting and repair. The lower panels show immunoblotting for the checkpoint kinase Rad53 from the same time course as the repair analysis, using a pan Rad53 antisera. The panels at the bottom show immunoblotting for phosphorylated Rad53 only. <b>D</b>. Calculation of colony formation from single unbudded cells following the indicated length of times of growth on galactose-containing plates in WT (YMV045), <i>rtt101</i>Δ (CCY019), <i>mms1</i>Δ (CCY018) and <i>asf1</i>Δ (JKT200) strains. Error bars represent standard deviation calculated from three independent experiments. <b>E</b>. Analysis of DNA levels (input) and H3 levels flanking the HO lesion using the identical strains shown in <b>D</b>. The left panel shows the input, and the right panel shows the ChIP analysis of H3. Both input and ChIP data were normalized as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180556#pone.0180556.g001" target="_blank">Fig 1C</a>. All data are the average and standard deviation of three independent experiments.</p

Yeast strains.

<p>Yeast strains.</p

CAF-1 but not Rtt106 is involved in chromatin assembly after DSB repair.

<p><b>A</b>. Experimental system for measuring SSA. The galactose inducible HO endonuclease system induces one specific DSB in a defined location (red arrow) in order to analyze the DSB repair <i>in vivo</i> by PCR analysis with the indicated primer pairs. Repair of the HO lesion at the HO cleavage site (blue box) requires 5kb of resection back to the uncleavable HO cleavage site (red box). <b>B</b>. 10-fold serial dilution analysis of the indicated isogenic yeast strains WT (YMV045), <i>asf1</i>Δ (JKT200), <i>rad52</i>Δ (YMV046), <i>cac2</i>Δ (JLY078), and <i>cac2</i>Δ <i>rtt106</i>Δ (CCY020) to show the sensitivity to a single HO lesion. The WT data in the top panel are from the same petri dish as the other strains, but sections of the photograph were rearranged to obtain an optimal order of the strains for the figure. <b>C</b>. ChIP analysis of H3 levels flanking the DSB site (0.6kb) in WT (YMV045), <i>asf1</i>Δ (JKT200) and <i>cac2</i>Δ (JLY078) strains. The HO lesion was induced by adding galactose at 0hr. The H3 ChIP data were normalized to a control region on another chromosome (<i>SMC2</i>). All data are the average and standard deviation of three independent experiments.</p

Blocking chromatin assembly leads to persistent checkpoint sensor presence at the site of repair.

<p><b>A</b>. The top panels show the histone H3 K56Ac levels assessed by ChIP analysis adjacent to the DSB site (0.6Kb) and at a control region <i>SMC2</i> in a wild type strain (YMV045). The H3 K56Ac ChIP data were normalized to H3 levels and to the input. All data are the average and standard deviation of three independent experiments. Asterisk (*) indicates significant changes compared to time 0 (p<0.05), as determined by the Student’s t-test. The lower panels show immunoblotting for the checkpoint kinase Rad53, where activation of the checkpoint leads to phosphorylation of Rad53 (Rad53-P) apparent as a slower migrating species. Tubulin serves as a loading control. Right panels (+Nicotinamide) are as in left panels, but in the presence of 25 mM nicotinamide. <b>B</b>. Analysis of proportion of cells with Ddc1-RFP in repair foci or <b>C</b>. Ddc2-GFP in repair foci at the indicated times following addition of galactose. Over 100 cells were examined at each time point. The data shown are from one experiment and are representative of the typical results.</p

Mms22 promotes DSB repair via loading of Rad51.

<p><b>A</b>. Analysis of stability of Mms22-HA at the indicated times after shutting off transcription of pGAL1-Mms22HA by addition of glucose to cultures previously grown in YEPG. Strains used are wild type pGAL1-Mms22HA (LWY016), <i>asf1</i>Δ (CFY039) and <i>rtt101</i>Δ (CFY046). <b>B</b>. The HO endonuclease was induced by addition of galactose at 0hr in the WT (YMV045) and <i>mms22</i>Δ (CCY024) strains. The top panels show an analysis of the cutting and repair. The lower panels show immunoblotting for the checkpoint kinase Rad53 from the same time course as the repair analysis. <b>C</b>. ChIP analysis of Rad51 levels flanking the DSB site and a TELVIR control region in WT (YMV045) and <i>mms22</i>Δ (CCY024) strain. Data are normalized to the input. Data are the average and standard deviation of three independent experiments.</p