| Brc1 is a multi-BRCT domain protein in Schizosaccharomyces pombe that is required for resistance to replicative stress and the viability of Smc5/6 hypomorphs, while overexpression of Brc1 rescues the DNA damage sensitivity of most Smc5/6 hypomorphs. Brc1 is homologous to Rtt107 in Saccharomyces cerevisiae, which is implicated in replication restart following DNA damage. Here, I show that Brc1 is important for regulated replication fork stalling, and also for the efficient restart of stalled replication forks. I find that Brc1 is required for the recruitment of Rad52 to stalled replication forks early in S-phase, which sequesters it from forming repair foci following DNA damage in checkpoint-competent cells that have undergone replication arrest. When this response is defective, the free Rad52 in S-phase arrested cells is competent to form foci despite intact intra-S-phase checkpoint signaling, but does not protect cells from replicative damage. Additionally, this defect correlates with impaired recombination between repeat sequences in cells lacking brc1. I further show that brc1Delta cells accumulate Rad52 foci late in S-phase, which are substantially enhanced by a prior replication arrest. These foci do not contain Rad51, as seen with DNA damage, but rather contain Replication Protein A and are associated with a delayed recovery from replication stalling. Brc1 binds chromatin in an unperturbed S-phase, but disassociates from chromatin when replication stalls, dependent on the intra-S-phase checkpoint and the Smc5/6-dependent regulation of stalled replication forks. Slx1, a structure-specific nuclease required for Brc1 to suppress smc6-74, localizes to chromatin with similar kinetics and in a Brc1-dependent manner. Brc1 promotes mutagenesis, in large part dependent on bypass polymerases, which are also required for Brc1-mediated suppression of smc6-74. Thus, I propose that Brc1 contributes to the establishment of proper replication fork architecture following replication stalling, as well as the efficient recovery from replication arrest in an error-prone manner via bypass polymerases and nucleases that cleave stalled forks. My findings provide mechanisms by which this conserved protein protects cells from replicative stress, and thereby provide protection from genome instability. |
