Recovery of Arrested Replication Forks by Homologous Recombination Is Error-Prone

Abstract

Homologous recombination is a universal mechanism that allows repair of DNA and provides support for DNA replication. Homologous recombination is therefore a major pathway that suppresses non-homology-mediated genome instability. Here, we report that recovery of impeded replication forks by homologous recombination is error-prone. Using a fork-arrest-based assay in fission yeast, we demonstrate that a single collapsed fork can cause mutations and large-scale genomic changes, including deletions and translocations. Fork-arrest-induced gross chromosomal rearrangements are mediated by inappropriate ectopic recombination events at the site of collapsed forks. Inverted repeats near the site of fork collapse stimulate large-scale genomic changes up to 1,500 times over spontaneous events. We also show that the high accuracy of DNA replication during S-phase is impaired by impediments to fork progression, since fork-arrest-induced mutation is due to erroneous DNA synthesis during recovery of replication forks. The mutations caused are small insertions/duplications between short tandem repeats (micro-homology) indicative of replication slippage. Our data establish that collapsed forks, but not stalled forks, recovered by homologous recombination are prone to replication slippage. The inaccuracy of DNA synthesis does not rely on PCNA ubiquitination or trans-lesion-synthesis DNA polymerases, and it is not counteracted by mismatch repair. We propose that deletions/insertions, mediated by micro-homology, leading to copy number variations during replication stress may arise by progression of error-prone replication forks restarted by homologous recombination.

Characterization of 26 deletion CNVs reveals the frequent occurrence of micro-mutations within the breakpoint-flanking regions and frequent repair of double-strand breaks by templated insertions derived from remote genomic regions

“…The increased mutational complexity included micro-deletions and micro-insertions, apparently originating from DNA polymerase slippage events at the breakpoint junctions (29 %), and small frameshifts or point mutations in homonucleotide runs within the breakpointflanking sequences (13 %; distance to the breakpoints was <45 bp). This increase in mutational complexity has been attributed to the activity of low-fidelity, error-prone DNA polymerases used during DNA repair via replication-based mechanisms (Carvalho et al 2013), a conclusion supported by multiple previous studies (Arlt et al 2012;Deem et al 2011;Hicks et al 2010;Iraqui et al 2012;Shah et al 2012;Smith et al 2007). Somewhat unexpectedly, all three of our disease-causing deletion CNVs, when characterized at the nucleotide level, were found to have cis-linked additional mutations.…”

Regulation of recombination at yeast nuclear pores controls repair and triplet repeat stability

“…For example, a recent study showed that Sgs1 foci are reduced after HU treatment in a Slx8-dependent manner (Bohm et al 2015). At a protein-mediated stall, the restarted fork was more prone to replication slippage, producing deletions (Iraqui et al 2012). A similar fork restart mechanism could be occurring at the CAG repeat, contributing to the low level of instability, biased toward contractions, that is observed in unperturbed wild-type cells (Fig.…”

Repeat instability during DNA repair: Insights from model systems

“…Recent results show that replication in the context of HR repair or HR-dependent fork restart proceeds with less fidelity and more mutations than normal replication, even without the complication of copying DNA repeats (Deem et al ., 2011; Hicks et al ., 2010; Iraqui et al ., 2012). Recently, GAA repeats have been shown to induce mutagenesis up to 8 kb from the repeat site in yeast, which is enhanced in strains with polymerase defects and largely dependent on the Polζ TLS polymerase (Saini et al ., 2013; Shah et al ., 2012; Tang et al ., 2013).…”