RAD51 and MRE11 dependent reassembly of uncoupled CMG helicase complex at collapsed replication forks

In higher eukaryotes the dynamics of replisome components during fork collapse and restart are poorly understood. Here, we reconstituted replication fork collapse and restart by inducing single-strand DNA (ssDNA) lesions that create a double-strand break (DSB) in one of the replicated sister chromatids after fork passage. We found that, upon fork collapse, the active CDC45–MCM–GINS (CMG) helicase complex loses its GINS subunit. A functional replisome is restored by the reloading of GINS and Pol epsilon onto DNA in a RAD51- and MRE11- dependent manner, but independently of replication origin assembly and firing. PCNA mutant alleles defective in break-induced replication (BIR) are unable to support restoration of replisome integrity. These results reveal that in higher eukaryotes replisomes are partially dismantled following fork collapse and fully re-established by a recombination-mediated process.