Using conditional fork arrest constructs, we studied the consequences for genome instability of impediments to replication forks progression. A single fork arrest results in large-scale genomic changes and mutations that occur during recombination-mediated fork recovery (). Inappropriate ectopic recombination at arrested forks results in GCRs, whereas appropriate restarting of the fork on the initial template results in error-prone DNA synthesis. GCRs and mutations at collapsed forks are genetically separable: Rqh1 limits fork-arrest-induced GCRs but not mutations ( and ). We demonstrate here that collapsed forks whose progression resumes by recombination lose accuracy during DNA synthesis, resulting in frequent intra-template switches. Thus, homologous recombination contributes to completion of DNA replication when forks progression is impeded but also fuels genome modifications both at the chromosomal and nucleotide level.
Model of replication-stress-induced genetic instability at collapsed forks.
Non allelic homologous recombination (NAHR) between low copy number repeats (LCR) contributes to recombination-mediated GCRs in mitosis and meiosis. NAHR is responsible for translocations, deletions, inversions and loss of heterozygosity 
. Ou et al.
predicted 1143 LCR pairs in the human genome liable to mediate recurrent translocations 
. In budding yeast, a single DSB is sufficient to mediate recombination-dependent translocation 
. Here, we report that a single collapsed fork increases the rate of genomic deletion 40 fold, and that of translocation 5 fold. Fork-arrest-induced GCRs are mediated by NAHR between heterologous chromosomes. It is not clear whether fork arrest on both homologous repeats contributes to fork-arrest-induced GCRs. We could not address this question in our model system, because the RTS1
sequence close to the mat1
locus on chromosome II has a low RFB activity 
. Also, the recruitment of recombination proteins at the RTS1
sequence near the mat1
locus is not regulated by the level of Rtf1 expression, showing that regulating Rtf1 expression was in itself insufficient to regulate the RTS1
-RFB activity at the mat1
Inverted repeats (IRs) are structural elements that contribute to genome instability. Impediments to replication forks progressing through IRs favour their fusion to generate acentric and dicentric inverted chromosomes 
. IRs in humans can also trigger the formation of inverted genomic segments and complex triplication rearrangements by a replication-based mechanism 
. Here, we report that IRs near collapsed forks can increase the rate of GCRs by up to 1,500 fold. This high level of GCRs cannot be explained merely by the addition of independent inter- and intra-chromosomal recombination events. Rather, our analyses suggest that IRs may stimulate tri-parental recombination events induced by template switching of nascent strands at collapsed forks, such that three homologous sequences are involved. Similarly, recombination-dependent translocations induced by a single DSB in budding yeast is proposed to be the consequence of tri-parental recombination 
. One possible mechanism is that IRs-induced GCRs result from successive template switches initiated by nascent strands at collapsed forks, reminiscent of the multiple template switches during break-induced-replication (BIR) in budding yeast 
. Interestingly, Rqh1 prevents fork-arrest-induced GCRs by limiting both inter- and intra-chromosomal recombination without affecting fork restart efficiency. Thus, tri-parental recombination might correspond to multiple and successive template switches between homologous repeats.
The high accuracy of DNA replication is compromised by impediments to fork progression, and recombination-mediated fork recovery results in decreased processivity of DNA synthesis (). Recombination-induced mutations associated with DSBs or impeding DNA replication have been described previously. The formation of damaged single-stranded DNA during the resection of DSBs favours base-substitutions 
. We detected the recruitment of the single-strand binding protein RPA up to 1.4 kb behind, but not ahead of the RTS1
-arrested fork (data to be published), showing that fork-arrest-induced mutation is not correlated with damaged single-stranded DNA exposed behind collapsed forks. Nevertheless, there were rare replication slippage occurred behind RTS1
-arrested forks (in the t<ura4-ori
construct), suggesting that resumption of DNA synthesis can in some cases occur at a position behind the site of the collapsed fork. Recombination-dependent DNA synthesis occurring outside S-phase is also highly inaccurate during gene conversion and BIR, resulting in either template switches, base-substitutions or frame-shifts 
. Elevated dNTP pools, due to activation of the DNA-damage checkpoint in G2 cells, contributes to the generation of mutations when hundreds of kbs are synthesised during BIR 
. Here, we demonstrate that during normal S-phase progression, a single collapsed fork, restored by recombination, results in replication slippage up to 1.2 Kb away from the initial restarting point.
Recombination-induced replication slippage has been reported previously.
In fission yeast, a defect in pol alpha (swi7-H4
mutant) is associated with a recombination-mediated mutator phenotype characterized by an increased frequency of base-substitutions and Del/Dup between short direct repeats 
. DNA-polymerase kappa (DinB) and zeta (Rev3) are responsible for the increased base-substitution rate, but the DNA-polymerases involved in Del/Dup mutations were not identified 
. In budding yeast, a defect in polymerase delta (pol3-t
mutant) results in an increased level of replication slippage, mediated by homologous recombination 
. In contrast, the increased rate of replication slippage in the absence of the accessory subunit of polymerase delta, Pol32, does not depend on a functional recombination pathway 
. Here, we report that recovery of collapsed forks by recombination is specifically associated with replication slippage. Nonetheless, spontaneous replication slippage events are also increased in strains mutated for recombination genes (). Pol32 is required for BIR and replication-induced template switches leading to segmental duplication 
. Recombination is responsible for only half of these segmental duplications. Thus, it is possible that fork-restart mechanisms dependent on Pol32 and homologous recombination are prone to replication slippage and that in the absence of these pathways alternative micro-homology mediated mechanisms are revealed.
We suggest that at least two steps of the recombination-based fork recovery mechanism can compromise genome stability (). At an initial stage, recruitment of recombination proteins on stalled nascent strands favours both fork recovery and ectopic template switches leading to GCRs. At a later stage, once the replisome has been reconstituted and the fork has resumed its progression, the nascent strands are prone to intra-template switching leading to replication slippage. The reasons for the inaccuracy of DNA synthesis associated with restarted forks during scheduled DNA replication (i.e.
in S-phase) remain to be determined. One possibility is that one or more factors are missing in the rebuilt replisome during recovery by recombination. Oncogene-induced replication stress results from unbalanced DNA replication that contributes to genome instability in precancerous cells 
. Completion of DNA replication in such stress conditions is likely to rely on recombination-mediated fork recovery that in turn generates genome instability. Insertions/deletions flanked by micro-homology, responsible for copy number variations (CNVs), have been identified both in cancer cells and also in response to replication inhibition 
; their reported sizes are between 1 Kb and several tens of mega bases, but the analysis of these features has been limited by the resolution of array-based genomics approaches. Sub-microscopic insertions/deletions flanked by micro-homology have been also described at loci in which replication origins are scarce, including the human fragile site FRA3B, the instability of which is a consequence of replication stress 
. Interestingly, homologous recombination contributes to the stability of fragile sites by facilitating complete replication or by repairing gaps and breaks at these sites. Thus, we propose that micro-homology-mediated CNVs could be viewed as scars left by error-prone replication forks restarted by recombination.