2017
DOI: 10.1038/nature22046
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Rad51-mediated double-strand break repair and mismatch correction of divergent substrates

Abstract: The RecA/Rad51 family of recombinases execute the critical step in homologous recombination (HR): the search for homologous DNA to serve as the template during DNA double-strand break (DSB) repair1–7. Although budding yeast Rad51 has been extensively characterized in vitro3,4,6–9, the stringency of its search and sensitivity to mismatched sequences in vivo remain poorly defined. We analyzed Rad51-dependent break-induced replication (BIR) where the invading DSB end and its donor template share 108 bp homology a… Show more

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Cited by 135 publications
(172 citation statements)
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“…Initiation by Pol δ, however, does not exclude subsequent recruitment of Pol ε or of a translesion synthesis DNAP to bypass obtacles in the repair template (Rattray et al, 2002; Roberts et al, 2012). Consistent with our observations of single-SNP removal on both sides of the initiating break, removal of a terminal SNP located 9 nt from an invading 3′ end was recently reported in a BIR assay (Anand et al, 2017). In terms of how far Pol δ-mediated excision might normally extend during DSB repair, we note that correction of a mismatch located 18 bp from the end did not occur in a plasmid-based assay (Mitchel et al, 2010).…”
Section: Discussionsupporting
confidence: 93%
“…Initiation by Pol δ, however, does not exclude subsequent recruitment of Pol ε or of a translesion synthesis DNAP to bypass obtacles in the repair template (Rattray et al, 2002; Roberts et al, 2012). Consistent with our observations of single-SNP removal on both sides of the initiating break, removal of a terminal SNP located 9 nt from an invading 3′ end was recently reported in a BIR assay (Anand et al, 2017). In terms of how far Pol δ-mediated excision might normally extend during DSB repair, we note that correction of a mismatch located 18 bp from the end did not occur in a plasmid-based assay (Mitchel et al, 2010).…”
Section: Discussionsupporting
confidence: 93%
“…Although we observed a reduction in 3′ bias with a PAGE purified ssODN (Figure S3D), this effect was not completely eliminated with PAGE purification, suggesting that the effect could be a combination of truncated ssODNs from synthesis and native processing. The greater read-depth with HTS allowed us to uncover additional processing events at the 3′, potentially due to proofreading activity of DNA-polymerase δ (Anand et al, 2017), and in some cases we observed clones lacking an internal mutation in the ssODN but retaining the 5′ and 3′ mutations (Figure 4G–I). …”
Section: Resultsmentioning
confidence: 99%
“…As noted above, a BIR assay using 108-bp substrates showed that Rad51 has tolerance for a substantial degree of mismatching [97]. This study of BIR and sequence divergence revealed another unexpected finding: the presence of a nonhomologous sequence at the 3’ end of the invading strand (which is readily clipped off by Rad1-Rad10 [98]) dramatically changed the sensitivity of the system to mismatches; now, even a single mismatch was sufficient to reduce repair by a factor of two [97].…”
Section: How Does the Search For Homology Differ From A Search For Homentioning
confidence: 99%
“…This study of BIR and sequence divergence revealed another unexpected finding: the presence of a nonhomologous sequence at the 3’ end of the invading strand (which is readily clipped off by Rad1-Rad10 [98]) dramatically changed the sensitivity of the system to mismatches; now, even a single mismatch was sufficient to reduce repair by a factor of two [97]. A similar finding has been made concerning SSA (E. Sapede, N. Sugawara and JEH, unpublished).…”
Section: How Does the Search For Homology Differ From A Search For Homentioning
confidence: 99%