Functional Interplay between the 53BP1-Ortholog Rad9 and the Mre11 Complex Regulates Resection, End-Tethering and Repair of a Double-Strand Break

<div><p>The Mre11-Rad50-Xrs2 nuclease complex, together with Sae2, initiates the 5′-to-3′ resection of Double-Strand DNA Breaks (DSBs). Extended 3′ single stranded DNA filaments can be exposed from a DSB through the redundant activities of the Exo1 nuclease and the Dna2 nuclease with the Sgs1 helicase. In the absence of Sae2, Mre11 binding to a DSB is prolonged, the two DNA ends cannot be kept tethered, and the DSB is not efficiently repaired. Here we show that deletion of the yeast 53BP1-ortholog <i>RAD9</i> reduces Mre11 binding to a DSB, leading to Rad52 recruitment and efficient DSB end-tethering, through an Sgs1-dependent mechanism. As a consequence, deletion of <i>RAD9</i> restores DSB repair either in absence of Sae2 or in presence of a nuclease defective MRX complex. We propose that, in cells lacking Sae2, Rad9/53BP1 contributes to keep Mre11 bound to a persistent DSB, protecting it from extensive DNA end resection, which may lead to potentially deleterious DNA deletions and genome rearrangements.</p></div

Deletion of <i>RAD9</i> rescues <i>sae2</i>Δ and <i>mre11</i>-D56N cell viability following DSBs through <i>SGS1</i>.

<p>(A–B) Viability of the wild type YMV80 strain and the indicated derivatives plated on YEP+gal. In the presence of galactose, one HO-cut is introduced at <i>leu2</i> locus (see a scheme in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004928#pgen-1004928-g002" target="_blank">Fig. 2A</a>). For each strain, the number of colonies grown after 3 days at 28°C in YEP+gal was normalized respect YEP+glu. Plotted values are the mean values ± SD from three independent experiments. (C) Exponentially growing cell cultures of the wild type YMV80 strain and the indicated derivatives were serially diluted (1∶10), and each dilution was spotted out into YPD and YPD+camptothecin plates. Plates were incubated 3 days at 28°C.</p

Rad9 limits an Sgs1- and Sae2- dependent initial step of DSB resection.

<p>(A) Scheme of the <i>MAT</i> locus. The figure shows the positions of the HO-cut site, and the probe used in experiments shown in (B and C) and in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004928#pgen.1004928.s003" target="_blank">S3</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004928#pgen.1004928.s004" target="_blank">S4 Figs</a>. (B, C) Exponentially growing YEP+raf cell cultures of the wild type JKM139 strain and the indicated derivatives, carrying a unique HO cut site at <i>MAT</i> locus and expressing the HO nuclease under GAL1 promoter, were synchronized and kept in G2/M phases by nocodazole treatment. Galactose was added at time 0 to induce HO. SspI-digested genomic DNA, extracted from samples taken at the indicated times, was analysed by Southern blotting to test 3′ filament formation. (C) The mean values ± SEM corresponding to the resection products of two independent experiments were determined by densitometry. (D) Schematic representation of the quantitative PCR method used to monitor HO-induced DSB resection. (E–F) Plots showing the ratio of resected DNA among HO cut DNAs at each time points by qPCR analysis. The mean values from three independent experiments are shown with SEM. Significance was calculated by one-tailed paired Student's <i>t</i> test (* for P<0.05; ** for P<0.01; where not indicated, the P value was higher than 0.05) (G) JKM139 derivatives were nocodazole-arrested in G2/M and 2% galactose was added to induce HO cut. After 2 hours of HO induction, cells were plated on YEP+raf and YEP+raf+gal, and incubated at 28°C for three days. Viability results were obtained from the ratio between number of colonies on YEP+raf+gal and YEP+raf. The mean values from three independent experiments are shown with SD.</p

Deletion of <i>RAD9</i> rescues DSB repair defects of <i>sae2</i>Δ cells through <i>SGS1</i> and <i>DNA2</i>.

<p>(A) Map of the YMV80 Chr III region, containing the HO-cut site. The indicated vertical bars show KpnI restriction sites. The short thick lines indicate the position where the probe hybridizes. After the HO mediated cleavage, DNA ends are resected. Once the indicated <i>leu2</i> cassettes have been exposed as ssDNA, repair through SSA can occur and be monitored by the appearance of an SSA product fragment by Southern blot. (B and D) Exponentially growing YEP+raf cell cultures of the wild type YMV80 strain and the indicated derivatives were synchronized and kept blocked in G2/M phase with nocodazole treatment; galactose was added at time zero to induce HO-cut. <i>KpnI</i>-digested DNA was analysed by Southern blotting with a <i>LEU2</i> probe. An <i>ATG5</i> (uncut locus on chromosome XVI) probe was also used to normalize the signals. In (D) <i>LEU2</i> and <i>ATG5</i> probes were added contemporarily to the filter. (C and E) Densitometric analysis of the product band signals of the experiments shown in (B) and (D). The intensity of each band was normalized respect to unprocessed <i>ATG5</i> locus (*).</p

Rad9 oligomers affect cell viability following a DSB, in the absence of Sae2, mainly through the interaction with Dpb11.

<p>(A and D) Viability of the wild type YMV80 strain and the indicated derivatives, plated on YEP+raf+gal. For each strain, the number of colonies grown after 3 days at 28°C in YEP+raf+gal was normalized respect YEP+raf. Plotted values are the mean values ± SD from three independent experiments. (B) Cells of the wild type JKM139 strain and the indicated derivatives, expressing a Rad9-3HA fusion protein, were grown in YEP+raf and synchronized in G2/M phases by nocodazole treatment. Galactose was added at time 0 to induce HO. Relative fold enrichment of Rad9-3HA at 0.1 kb from the HO cleavage site was evaluated after ChIP with anti-HA antibodies and qPCR analysis. Plotted values are the mean values ± SEM from three independent experiments. (C) Schematic representation of Rad9 functional domains and sites phosphorylated by CDK1, Mec1 and Tel1. (E) Exponentially growing cell cultures of the wild type YMV80 strain and the indicated derivatives were incubated for 2 hours with or without the dimerization-inducing molecule AP20187, before plating in YEP+Raf or YEP+Raf+Gal, with/without AP20187. For each strain, the number of colonies grown after 3 days at 28°C in YEP+raf+gal was normalized with respect to YEP+raf. Plotted values are the mean values ± SD from three independent experiments. Expression level of Rad9-2A, Rad9-7xA and Rad9-ΔBRCT-FKBP protein variants, described in this Figure, were determined by western blotting in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004928#pgen.1004928.s006" target="_blank">S6 Fig</a>.</p

Model to explain the interplay between Mre11 complex and Rad9 at a DSB in G2/M phase.

<p>Ku and Mre11 complexes, together with Rad9, are recruited soon after a DSB formation and limit the action of Exo1 and Dna2-Sgs1 pathways. The order of appearance of the various factors was based on both literature and our results. See details in the text.</p

Rad9 limits Mre11 removal from a DSB, affecting Rad52 binding and DSB ends tethering in <i>sae2</i>Δ cells.

<p>(A, B) Cells of the wild type JKM139 strain and the indicated derivatives, expressing a Mre11–18Myc fusion protein, were grown in YEP+raf and synchronized in G2/M phases by nocodazole treatment. Galactose was added at time 0 to induce HO. Relative fold enrichment of Mre11–18Myc at 0.1 kb from the HO cleavage site was evaluated after ChIP with anti-Myc antibodies and qPCR analysis. Plotted values are the mean values ± SEM from three independent experiments. (C) Cells of the wild type JKM139 strain and the indicated derivatives, expressing a Rad52-RFP fusion protein, were grown in YEP+raf and synchronized in G2/M phases by nocodazole treatment. Galactose was added at time 0 to induce HO. After 6 hours from DSB, cells were imaged under live cell conditions for Rad52-RFP focus formation. Approximately 100 cells per experiment were analyzed and the percentage of cells displaying a detectable Rad52-RFP focus was quantitated. Error bars reflect ranges from two independent experiments. (D) Cells of the wild type yJK40.6 strain and the indicated derivatives, expressing a LacI-GFP and carrying two <i>LacO</i> arrays (green boxes) at 50 kb on either side of one HO cut site on chromosome VII (see a scheme above the graph in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004928#pgen-1004928-g004" target="_blank">Fig. 4D</a> and text for details), were grown in YEP+raf and blocked in G2/M phases by nocodazole treatment. Galactose was added at time 0 to induce HO. Cell samples taken at the indicated times after HO induction were analysed with a fluorescence microscope to determine the percentage of cells in each sample that contained two LacI-GFP foci separated by>0.5 µm. The separation distance between foci was measured for 200 cells/sample.</p