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Mechanisms restraining break‐induced replication at two‐ended DNA double‐strand breaks
Journal article   Open access   Peer reviewed

Mechanisms restraining break‐induced replication at two‐ended DNA double‐strand breaks

Nhung Pham, Zhenxin Yan, Yang Yu, Mosammat Faria Afreen, Anna Malkova, James E Haber and Grzegorz Ira
The EMBO journal, Vol.40(10), pp.e104847-n/a
05/17/2021
DOI: 10.15252/embj.2020104847
PMCID: PMC8126933
PMID: 33844333
url
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8126933View
Published (Version of record) Open Access

Abstract

DNA synthesis during homologous recombination is highly mutagenic and prone to template switches. Two‐ended DNA double‐strand breaks (DSBs) are usually repaired by gene conversion with a short patch of DNA synthesis, thus limiting the mutation load to the vicinity of the DSB. Single‐ended DSBs are repaired by break‐induced replication (BIR), which involves extensive and mutagenic DNA synthesis spanning up to hundreds of kilobases. It remains unknown how mutagenic BIR is suppressed at two‐ended DSBs. Here, we demonstrate that BIR is suppressed at two‐ended DSBs by proteins coordinating the usage of two ends of a DSB: (i) ssDNA annealing proteins Rad52 and Rad59 that promote second end capture, (ii) D‐loop unwinding helicase Mph1, and (iii) Mre11‐Rad50‐Xrs2 complex that promotes synchronous resection of two ends of a DSB. Finally, BIR is also suppressed when Sir2 silences a normally heterochromatic repair template. All of these proteins are particularly important for limiting BIR when recombination occurs between short repetitive sequences, emphasizing the significance of these mechanisms for species carrying many repetitive elements such as humans. Synopsis While mutagenic break‐induced replication (BIR) repairs single‐ended DNA double‐strand breaks (DSBs), it needs to be suppressed at two‐ended breaks to ensure their faithful repair via gene conversion. Here, the coordinated usage of both DSB ends by ssDNA annealing and resection pathways is found to restrain unwanted BIR in budding yeast. Rad52‐ and Rad59‐mediated ssDNA annealing suppresses BIR by promoting second‐end capture. The Mre11‐Rad50‐Xrs2 complex suppresses BIR by promoting the synchronous formation of ssDNA at both ends of a break. ssDNA annealing is less important for DSB repair and preventing BIR when homology is long. Repair by BIR is not equally initiated at different genomic loci. Coordinated usage of both DNA break ends via annealing proteins Rad52/59, D‐loop‐unwinding Mph1 helicase and the MRX complex ensures their repair by gene conversion, rather than by BIR operating at single‐ended breaks.
 break‐induced replication double‐strand break DSB end resection homologous recombination ssDNA annealing

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