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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Available from: John Lydeard, Oct 04, 2015
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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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    G3-Genes Genomes Genetics 05/2015; 5(7). DOI:10.1534/g3.115.018721 · 3.20 Impact Factor
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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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    Chromosoma 03/2013; 122(3). DOI:10.1007/s00412-013-0404-2 · 4.60 Impact Factor
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    • "In the absence of selection, the targeted mutation rate in an sgs1 exo1 double mutant increased two orders of magnitude at 0.3 kb and was eliminated at −8 kb. De novo telomere addition is known to be a dominant repair pathway in this background (41), and this is consistent with the asymmetric results observed. The mutation rate increase is likely because breaks that could be repaired error-free at KlURA3 using HR now must be repaired using de novo telomere addition, which always results in a mutation on the telomeric side of the array. "
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