Competing roles of DNA end resection and non-homologous end joining functions in the repair of replication-born double-strand breaks by sister-chromatid recombination

Muñoz-Galván, Sandra; López-Saavedra, Ana; Jackson, Stephen; Huertas, Pablo; Cortés‐Ledesma, Felipe; Aguilera, Andrés

doi:10.1093/nar/gks1274

Cited by 15 publications

(13 citation statements)

References 68 publications

(52 reference statements)

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“…As expected, we observed a deviation of the balance towards an increase of homology-mediated repair (Figure 4, Tables S2 and S3). These results agree with previously published reports that indicate that, in the absence of NHEJ, DNA-end are unprotected, resection occurs, and the breaks are more prone to be repaired by homologous recombination [5], [16]–[21]. Although we observed a similar trend for both SSR 1.0 and 2.0, it was clearer in the latter (Figure 4).…”

Section: Resultssupporting

confidence: 93%

New Tools to Study DNA Double-Strand Break Repair Pathway Choice

et al. 2013

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A broken DNA molecule is difficult to repair, highly mutagenic, and extremely cytotoxic. Such breaks can be repaired by homology-independent or homology-directed mechanisms. Little is known about the network that controls the repair pathway choice except that a licensing step for homology-mediated repair exists, called DNA-end resection. The choice between these two repair pathways is a key event for genomic stability maintenance, and an imbalance of the ratio is directly linked with human diseases, including cancer. Here we present novel reporters to study the balance between both repair options in human cells. In these systems, a double-strand break can be alternatively repaired by homology-independent or -dependent mechanisms, leading to the accumulation of distinct fluorescent proteins. These reporters thus allow the balance between both repair pathways to be analyzed in different experimental setups. We validated the reporters by analyzing the effect of protein downregulation of the DNA end resection and non-homologous end-joining pathways. Finally, we analyzed the role of the DNA damage response on double-strand break (DSB) repair mechanism selection. Our reporters could be used in the future to understand the roles of specific factors, whole pathways, or drugs in DSB repair pathway choice, or for genome-wide screening. Moreover, our findings can be applied to increase gene-targeting efficiency, making it a beneficial tool for a broad audience in the biological sciences.

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Section: Resultssupporting

confidence: 93%

New Tools to Study DNA Double-Strand Break Repair Pathway Choice

et al. 2013

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“…It is likely that these yku mutants exhibit reduced affinity for DNA ends, as do those described by Petrini and colleagues 40 . If the basis of suppression of sae2D sgs1Dmutant phenotype by yku mutations is to restore end resection by allowing Exo1 access to DSBs arising during DNA replication 36,37,40,41 , then the rescue should be dependent on HR. We found that deleting RAD52 reduced the size of the sae2D sgs1D yKu70D spore colonies ( Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

Sgs1 and Sae2 promote telomere replication by limiting accumulation of ssDNA

et al. 2014

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In budding yeast, DNA ends are processed by the consecutive action of MRX/Sae2 and two redundant pathways dependent on Sgs1/Dna2 and Exo1, and this processing is counteracted by Ku heterodimer. Here we show that DNA end resection by Sae2 and Sgs1 is dispensable for normal telomere maintenance by telomerase. Instead, these proteins facilitate telomere replication and limit the accumulation of single-strand DNA (ssDNA) at replication fork pause sites. Loss of Sae2 and Sgs1 drives selection for compensatory mutations, notably in Ku, which are responsible for abrupt telomere shortening in cells lacking Sae2 and Sgs1. In telomerase-negative cells, Sae2 and Sgs1 play non-overlapping roles in generating ssDNA at eroded telomeres and are required for the formation of type II survivors. Thus, although their primary function in telomerase-positive cells is to sustain DNA replication over the sites that are prone to fork pausing, Sae2 and Sgs1 contribute to telomere resection in telomerase-deficient cells.

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“…However, rpb1‐S751F was tolerated in both rad51∆ and pol32∆ backgrounds. As we have previously shown that Rad51 and Pol32 control two overlapping outcomes of Rad52‐dependent HR events (Moriel‐Carretero & Aguilera, ; Muñoz‐Galván et al , ), we tested whether rpb1‐S751F rad51∆ pol32∆ triple mutants were viable or not. Indeed, they were not, suggesting that these mutants accumulate replication‐born DNA breaks that cannot be repaired in the absence of Rad51 and Pol32.…”

Section: Resultsmentioning

confidence: 99%

RNA polymerase II contributes to preventing transcription‐mediated replication fork stalls

et al. 2014

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Transcription is a major contributor to genome instability. A main cause of transcription-associated instability relies on the capacity of transcription to stall replication. However, we know little of the possible role, if any, of the RNA polymerase (RNAP) in this process. Here, we analyzed 4 specific yeast RNAPII mutants that show different phenotypes of genetic instability including hyperrecombination, DNA damage sensitivity and/or a strong dependency on double-strand break repair functions for viability. Three specific alleles of the RNAPII core, rpb1-1, rpb1-S751F and rpb9Δ, cause a defect in replication fork progression, compensated for by additional origin firing, as the main action responsible for instability. The transcription elongation defects of rpb1-S751F and rpb9Δ plus our observation that rpb1-1 causes RNAPII retention on chromatin suggest that RNAPII could participate in facilitating fork progression upon a transcription-replication encounter. Our results imply that the RNAPII or ancillary factors actively help prevent transcription-associated genome instability.

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Competing roles of DNA end resection and non-homologous end joining functions in the repair of replication-born double-strand breaks by sister-chromatid recombination

Cited by 15 publications

References 68 publications

New Tools to Study DNA Double-Strand Break Repair Pathway Choice

New Tools to Study DNA Double-Strand Break Repair Pathway Choice

Sgs1 and Sae2 promote telomere replication by limiting accumulation of ssDNA

RNA polymerase II contributes to preventing transcription‐mediated replication fork stalls

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