Eukaryotic cells rely on several mechanisms to ensure that the genome is duplicated precisely once in each cell division cycle, preventing DNA over-replication and genomic instability. Most of these mechanisms limit the activity of origin licensing proteins to prevent the reactivation of origins that have already been used. Here, we have investigated whether additional controls restrict the extension of re-replicated DNA in the event of origin re-activation. In a genetic screening in cells forced to re-activate origins, we found that re-replication is limited by RAD51 and enhanced by FBH1, a RAD51 antagonist. In the presence of chromatin-bound RAD51, forks stemming from re-fired origins are slowed down, leading to frequent events of fork reversal. Eventual re-initiation of DNA synthesis mediated by PRIMPOL creates ssDNA gaps that facilitate the partial elimination of re-duplicated DNA by MRE11 exonuclease. In the absence of RAD51, these controls are abrogated and re-replication forks progress much longer than in normal conditions. Our study uncovers a safeguard mechanism to protect genome stability in the event of origin reactivation.
Synopsis: Several mechanisms prevent DNA over-replication in eukaryotic cells. Here, a genetic screen reveals an additional role for RAD51 in slowing down and reversal of re-replication forks in the event of origin re-firing.RAD51 protein bound to newly replicated DNA hinders re-replication when origins are re-activated.Re-replicated forks undergo frequent fork reversal and rely on PRIMPOL-mediated repriming to maintain DNA synthesis.PRIMPOL-generated ssDNA gaps allow MRE11 exonuclease to access and degrade re-duplicated DNA.The RAD51-PRIMPOL-MRE11 axis serves as a safeguard against DNA over-replication.
RAD51-dependent slow-down and reversal of re-replication forks makes them susceptible to processing via PRIMPOL and MRE11, thereby protecting genome integrity upon origin re-firing.