Abstract A06: Regulation of homologous recombination by the SUMO ligase NSMCE2

Abstract

Abstract Introduction: The purpose of this study is to determine the role of the E3 SUMO ligase NSMCE2 in BLM regulation and homologous recombination (HR). Background: DNA damage generated during replication is a major source of mutations and failure to repair this damage can cause genomic instability. HR is a high-fidelity DNA repair pathway activated primarily by replication-associated DNA damage. Numerous HR proteins are regulated by sumoylation, but the mechanisms that control sumoylation and their roles in HR are poorly understood. Cells deficient in the SUMO E3 ligase NSMCE2 are sensitive to DNA damaging agents and have defects in HR. Our preliminary data indicated that BLM sumoylation is dependent on NSMCE2. Because BLM sumolyation is required to recruit RAD51 to stalled forks, we hypothesized that NSMCE2-deficient cells are deficient in HR due to a defect in BLM-dependent RAD51 recruitment. Results: To test this hypothesis, we transfected HeLa cells with siRNAs specific to NSMCE2 and tested whether cells could recruit RAD51 to replication forks stalled with hydroxyurea (HU). Contrary to our hypothesis, we found that the amount of RAD51 protein that accumulated at stalled forks was greater in HU-treated NSMCE2-deficient cells compared to HU-treated control cells. To our surprise, the amount of single-stranded DNA binding protein RPA was diminished by half in HU-treated NSMCE2-deficient cells and DNA damage signaling was similarly diminished as evidenced by the lower levels of γ-H2AX. Consistent with the low levels of γ-H2AX, the double-strand breaks (DSB) that normally form after extended treatment with HU were also greatly diminished in NSMCE2-deficient cells. Consistent with previous reports, we found that the levels of HU-induced sister chromatid exchange were also low. These data indicated that despite the over-accumulation of RAD51 to sites of stalled replication forks, cells are unable to perform HR repair efficiently, indicating that RAD51 is unable to complete its function there. Conclusions: The hyper-accumulation of RAD51 at stalled forks we observed in NSMCE2-deficient cells suggests the sumoylation of one or more substrates by NSMCE2 is required for the remodeling of stalled forks that normally leads to unloading of RAD51 at the fork, strand breakage, and repair by HR. We suggest that the RAD51 accumulation that is observed in a substantial fraction of cancers may not be sufficient to demonstrate that the cells are HR proficient. Based on our data, RPA staining combined with RAD51 may be able to distinguish HR-proficient and deficient cells. Citation Format: Kelvin W. Pond, Christelle DeRenty, Mary Yagle, Nathan Ellis. Regulation of homologous recombination by the SUMO ligase NSMCE2 [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A06.