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

Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts, United States of America.
PLoS Genetics (Impact Factor: 7.53). 05/2010; 6(5):e1000973. DOI: 10.1371/journal.pgen.1000973
Source: PubMed


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 sgs1Delta exo1Delta 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, sgs1Delta, or exo1Delta cells. In sgs1Delta exo1Delta, repair by GC is severely inhibited, but cell viability 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.

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    • "Consistent with this idea, an sgs1-D664∆ mutation, which is still competent in recombination repair but defective in resection[96,97], also prevents type II survivor formation[98]. Interestingly, extensive resection by Sgs1 and Exo1 inhibits Rad51-dependent BIR[99,100]. Thus, deletion of SGS1 or EXO1 may both promote the Rad51-dependent type I pathway and disrupt the Rad51-independent type II pathway. "

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    • "Additionally, like the delay in the sea3Δ Figure 2 Loss of Sea3 impacts colony formation in the break-induced replication (BIR) assay strain and on bleomycin. (A) BIR assay strain (Lydeard et al. 2010). An HO cut site (HO), marked with HPH, is integrated into the CAN1 gene (represented as CA) on chromosome V, deleting the 39 portion of CAN1. "
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    ABSTRACT: The Saccharomyces cerevisiae Iml1 complex inhibits TORC1 signaling and SEACAT antagonizes the Iml1 complex. Conditions in which SEACAT functions to inhibit Iml1 and, hence, TORC1 signaling, remain largely unknown. The SEACAT member Sea3 was linked previously to telomere maintenance and DNA repair via genome-wide genetic and physical interaction studies. Therefore, we questioned whether Sea3 functioned through TORC1 to influence these pathways. Deletion of SEA3 delayed emergence of telomerase independent survivors that utilize break-induced replication to maintain their telomeres. Similarly, sea3∆ mutants exhibited a delay in colony formation in a BIR assay strain following double strand break induction as well as on the DNA damaging agent bleomycin. Deletion of IML1 rescued the impaired growth of sea3∆ mutants following DNA damage, consistent with Sea3 functioning as a regulator of TORC1 signaling. The delay was not due to slowed DSB repair or termination of the DNA damage checkpoint, but due to tryptophan auxotrophy. High levels of tryptophan in yeast peptone dextrose media did not rescue the delay in colony formation, suggesting a defect in tryptophan import, though levels of the high affinity tryptophan permease Tat2 were not perturbed in the sea3∆ mutant. Addition of quinolinic acid, an intermediate of the de novo NAD+ biosynthetic pathway, however, rescued the delay in colony formation in the sea3∆ mutant. Together, these findings highlight the importance of enforcement of TORC1 signaling and suggest that internal tryptophan levels influence growth recovery post DNA damage through its role in NAD+ synthesis. Copyright © 2015 Author et al.
    Full-text · Article · May 2015 · G3-Genes Genomes Genetics
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    • "However, in strains defective for resection, Pif1 is still able to prevent telomere formation , suggesting that resection and Pif1 prevent telomere addition by two independent pathways. These data are supported by the fact that Cdc13 binding to a DSB is higher in pif1-m2 or exo1Δ sgs1Δ mutants and even higher in pif1-m2 exo1Δ sgs1Δ triple mutant cells (Chung et al. 2010; Lydeard et al. 2010). Altogether, these results indicate that impaired resection might stabilize Cdc13 binding and therefore promote telomere formation. "
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    ABSTRACT: Telomeres constitute the ends of linear eukaryotic chromosomes. Due to the conventional mode of DNA replication, telomeric DNA erodes at each cell division. To counteract this, a specialized reverse transcriptase, telomerase, can elongate chromosome ends to maintain them at a constant average length. Because of their similarity to DNA double-strand breaks (DSBs), telomeres might be expected to induce a DNA damage response, which would lead to repair reactions and the generation of translocations or fusions. Many proteins present at telomeres prevent this by protecting (capping) the chromosome termini. Conversely, a DSB occurring in other regions of the genome, due, for instance, to a stalled replication fork or genotoxic agents, must be repaired by homologous recombination or end-joining to ensure genome stability. Interestingly, telomerase is able to generate a telomere de novo at an accidental DSB, with potentially lethal consequences in haploid cells and, at a minimum, loss of heterozygosity (LOH) in diploid cells. Recent data suggest that telomerase is systematically recruited to DSBs but is prevented from acting in the absence of a minimal stretch of flanking telomere-repeat sequences. In this review, we will focus on the mechanisms that regulate telomere addition to DSBs.
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