Abstract 1356: NSMCE2 enables rescue of collapsed replication forks to prevent mitotic DNA damage

Abstract

Abstract Background: Replication forks that are persistently blocked cannot restart, even after DNA damage is repaired. These forks are termed collapsed forks. In order to fully duplicate the genome, collapsed forks must be rescued by activation of nearby dormant origins of replication. Homologous recombination (HR) is essential in collapsed-fork rescue. Cells deficient in the SUMO E3 ligase NSMCE2 exhibit mitotic defects, are sensitive to DNA damaging agents, and have defects in HR. Thus, NSMCE2 regulates HR during replication stress, but the molecular mechanisms are poorly understood. In yeasts, hypomorphic NSMCE2 (MMS21) alleles accumulate HR intermediates during replication stress. We therefore hypothesized that NSMCE2 regulates RAD51 function during collapsed fork rescue. Results: It was previously shown that sumoylation of the BLM helicase results in recruitment of RAD51 to stalled forks. Consistent with data in yeast, we found that BLM sumolyation is dependent on NSMCE2. However, contrary to our expectation, we found that the amount of RAD51 protein that accumulated at collapsed forks was over two times greater in NSMCE2-deficient cells than in normal cells. In contrast, the levels of BLM, RPA, single-stranded DNA, and γH2AX at stalled forks is reduced by half. Consistent with the low levels of γH2AX, the double-strand breaks and sister chromatid exchanges that accumulate during collapsed fork rescue were also greatly diminished in NSMCE2-deficient cells. Thus, despite the over-accumulation of RAD51 to sites of collapsed replication forks, cells are unable to perform HR efficiently, indicating that RAD51 is unable to complete its function there. In NSMCE2-deficient cells, the hyper-accumulated RAD51 at collapsed forks persists into mitosis where excess under-replicated DNA causes mitotic DNA damage. Conclusions: The hyper-accumulation of RAD51 at stalled forks we observed in NSMCE2-deficient cells suggests that NSMCE2 is required for the remodeling of collapsed forks that normally leads to the rescue of collapsed forks, namely, the unloading of RAD51, DNA breakage, repair by HR, and completion of DNA synthesis. We suggest that the excess accumulation of RAD51 that is observed in a substantial number of cancers is not sufficient to demonstrate that the cells are HR proficient. The identification of NSMCE2 as a controller of HR-mediated fork rescue also highlights NSMCE2's potential as a new therapeutic target for combinatorial therapy of HR-dependent cancers. Citation Format: Kelvin W. Pond, Christelle DeRenty, Mary K. Yagle, Nathan Ellis. NSMCE2 enables rescue of collapsed replication forks to prevent mitotic DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1356.