Inter-Fork Strand Annealing causes genomic deletions during the termination of DNA replication

Abstract

Problems that arise during DNA replication can drive genomic alterations that are instrumental in the development of cancers and many human genetic disorders. Replication fork barriers are a commonly encountered problem, which can cause fork collapse and act as hotspots for replication termination. Collapsed forks can be rescued by homologous recombination, which restarts replication. However, replication restart is relatively slow and, therefore, replication termination may frequently occur by an active fork converging on a collapsed fork. We find that this type of non-canonical fork convergence in fission yeast is prone to trigger deletions between repetitive DNA sequences via a mechanism we call Inter-Fork Strand Annealing (IFSA) that depends on the recombination proteins Rad52, Exo1 and Mus81, and is countered by the FANCM-related DNA helicase Fml1. Based on our findings, we propose that IFSA is a potential threat to genomic stability in eukaryotes.DOI: http://dx.doi.org/10.7554/eLife.25490.001

The PCNA unloader Elg1 promotes recombination at collapsed replication forks in fission yeast

“…To investigate whether Elg1 is required for IFSA, in the presence and absence of Rad51, we compared the frequency of RTS1-AO -induced recombination in wild-type, elg1 ∆, rad51 ∆ and rad51 ∆ elg1 ∆ strains containing the 0 kb site reporter with an extra 5 kb DNA spacer between the ade6 - repeats (Figure 6A,B). Consistent with our previous data (Morrow et al, 2017), the frequency of deletions increases ~10 fold in wild-type compared to the equivalent strain without a 5 kb spacer, whereas gene conversions increase by only ~1.5 fold. In an elg1 ∆ mutant the frequency of gene conversions decreases by ~19 fold, which is similar to the fold reduction seen in the equivalent strain without a 5 kb spacer (compare data in Figures 1D and 6B).…”

“…If Elg1 is required for efficient recruitment and/or retention of Rad52 to RTS1 , then it should be needed to promote Rad51-independent recombination at the barrier. We recently showed that Rad52 can promote strand annealing, between forks converging at RTS1 , independently of Rad51 (Morrow et al, 2017). This inter-fork strand annealing (IFSA) is enhanced when the spacing between the ade6 - repeats flanking RTS1 is increased.…”

Factors affecting template switch recombination associated with restarted DNA replication

“…Protocols for the growth and genetic manipulation of S. pombe , and assays for recombination have been described (Morrow et al, 2017; Nguyen et al, 2015). Between 3 and 10 colonies were assayed in each recombination experiment, with experiments repeated at least three times to achieve a minimum sample size as calculated using the Power calculation n = f ( α , β )(2 s 2 / δ 2 ) where α = 0.05; β = 0.1; s = 40; and δ = 50.…”

“…Replication forks collapse, and recruit HR proteins, within approximately 10 min of collision with RTS1 (Mohebi et al, 2015; Nguyen et al, 2015). Similar to BIR, Rad51 and Rad52 promote replication restart at RTS1 and, in doing so, can inadvertently cause genomic rearrangements through the recombination of repetitive DNA elements (Ahn et al, 2005; Lambert et al, 2005; Lambert et al, 2010; Mizuno et al, 2009; Morrow et al, 2017; Nguyen et al, 2015). Also like BIR, RDR induced by RTS1 is highly mutagenic and prone to TS (Iraqui et al, 2012; Mizuno et al, 2013; Nguyen et al, 2015).…”

Regulation of Structure-Specific Endonucleases in Replication Stress

“…Mus81 causes replication stress-induced double stranded breaks (DSB) in mammalian cells [38] and promotes deletion mutations in polα mutant fission yeast [44]. When an active replication fork converges on a collapsed fork, replication termination is prone to Mus81-dependent deletions between repetitive DNA sequences in fission yeast [45]. In human cells, oncogene-induced chromosomal breakage involves MUS81 activity [46].…”