Rescue and Repair of Collapsed Replication Forks

Abstract

NSMCE2 is an E3 SUMO ligase and a subunit of the SMC5/6 complex that associates with the replication fork and protects against genomic instability. Here, we study the fate of collapsed replication forks generated by prolonged hydroxyurea treatment in human NSMCE2-deficient cells. Double strand breaks accumulate during rescue by converging forks in normal cells but not in NSMCE2-deficient cells. Un-rescued forks persist into mitosis, leading to increased mitotic DNA damage. Excess RAD51 accumulates and persists at collapsed forks in NSMCE2-deficient cells, possibly due to lack of BLM recruitment to stalled forks. Despite failure of BLM to accumulate at stalled forks, NSMCE2-deficient cells exhibit lower levels of hydroxyurea-induced sister chromatid exchange. In cells deficient in both NSMCE2 and BLM, hydroxyurea-induced double strand breaks and sister chromatid exchange resembled levels found in NSCME2-deficient cells. We conclude that the rescue of collapsed forks by converging forks is dependent on NSMCE2. DNA damage encountered by the replication fork causes fork stalling and is a major source of mutations when not adequately repaired. Fork stalling can lead to fork collapse, that is, a state of the fork in which normal DNA synthesis cannot be resumed at the site of stalling. Collapsed forks must be rescued by replication forks initiated nearby, but little is known about the rescue mechanism by which an active fork merges with a collapsed fork. We used an inhibitor of DNA replication to generate collapsed replication forks and then studied genetic control of collapsed-fork rescue. We found that NSMCE2, which is a gene product that is known to regulate repair responses to replication stress, is required for cells to effectively rescue collapsed replication forks in order to complete DNA synthesis. DNA double strand breaks that are associated with normal collapsed-fork rescue do not accumulate in cells that are deficient for NSMCE2, suggesting that DNA breakage is part of the rescue and repair mechanism. Failure to rescue collapsed forks leads to DNA damage in mitosis and DNA damage in the following cell cycle. Our work highlights a unique role for NSMCE2 in rescue of collapsed replication forks. We then used the basic information about NSMCE2 and its role in the rescue of collapsed forks to generate the hypothesis that NSMCE2-deficient cells would reply more on topoisomerase proteins to help resolve excess topological stress. We used this to hypothesis to screen for potential NSMC2 inhibitors and discovered four potential compounds which sensitized a human cancer cell line to topoisomerase 1 poison. The finding that NSMCE2 inhibited cells rely more on topoisomerases has a clear impact. As an adjuvant therapy, NSMCE2 inhibition could significantly lower the effective dose of clinically approved, but highly toxic chemotherapies.