Sgs1 and Exo1 Redundantly Inhibit Break-Induced Replication and De Novo Telomere Addition at Broken Chromosome Ends

In budding yeast, an HO endonuclease-inducible double-strand break (DSB) is efficiently repaired by several homologous recombination (HR) pathways. In contrast to gene conversion (GC), where both ends of the DSB can recombine with the same template, break-induced replication (BIR) occurs when only the centromere-proximal end of the DSB can locate homologous sequences. Whereas GC results in a small patch of new DNA synthesis, BIR leads to a nonreciprocal translocation. The requirements for completing BIR are significantly different from those of GC, but both processes require 5′ to 3′ resection of DSB ends to create single-stranded DNA that leads to formation of a Rad51 filament required to initiate HR. Resection proceeds by two pathways dependent on Exo1 or the BLM homolog, Sgs1. We report that Exo1 and Sgs1 each inhibit BIR but have little effect on GC, while overexpression of either protein severely inhibits BIR. In contrast, overexpression of Rad51 markedly increases the efficiency of BIR, again with little effect on GC. In sgs1Δ exo1Δ strains, where there is little 5′ to 3′ resection, the level of BIR is not different from either single mutant; surprisingly, there is a two-fold increase in cell viability after HO induction whereby 40% of all cells survive by formation of a new telomere within a few kb of the site of DNA cleavage. De novo telomere addition is rare in wild-type, sgs1Δ, or exo1Δ cells. In sgs1Δ exo1Δ, repair by GC is severely inhibited, but cell viaiblity remains high because of new telomere formation. These data suggest that the extensive 5′ to 3′ resection that occurs before the initiation of new DNA synthesis in BIR may prevent efficient maintenance of a Rad51 filament near the DSB end. The severe constraint on 5′ to 3′ resection, which also abrogates activation of the Mec1-dependent DNA damage checkpoint, permits an unprecedented level of new telomere addition

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

A pathway of targeted autophagy is induced by DNA damage in budding yeast

Autophagy plays a central role in the DNA damage response (DDR) by controlling the levels of various DNA repair and checkpoint proteins; however, how the DDR communicates with the autophagy pathway remains unknown. Using budding yeast, we demonstrate that global genotoxic damage or even a single unrepaired double-strand break (DSB) initiates a previously undescribed and selective pathway of autophagy that we term genotoxin-induced targeted autophagy (GTA). GTA requires the action primarily of Mec1/ATR and Rad53/CHEK2 checkpoint kinases, in part via transcriptional up-regulation of central autophagy proteins. GTA is distinct from starvation-induced autophagy. GTA requires Atg11, a central component of the selective autophagy machinery, but is different from previously described autophagy pathways. By screening a collection of ∼6,000 yeast mutants, we identified genes that control GTA but do not significantly affect rapamycin-induced autophagy. Overall, our findings establish a pathway of autophagy specific to the DNA damage response

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