Abstract 3036: The SUMO-targeted ubiquitin ligase RNF4 regulates BLM helicase's function in dormant origin firing

Abstract

Abstract Regulation of dormant origin firing under conditions of replication stress is poorly understood. The Bloom syndrome DNA helicase BLM functions in maintaining replication fork stability, with sumoylation regulating this function, and BLM deficiency is associated with increased dormant origin firing. We found here that BLM sumoylation levels increase in response to replication stalling by hydroxyurea (HU) and to proteasome inhibition; depletion of the SUMO-targeted ubiquitin ligase RNF4 also causes increased levels of BLM sumoylation. RNF4 directly interacts with BLM and can ubiquitylate sumoylated BLM in vitro. These data indicate that sumoylated BLM is an RNF4 substrate. RNF4 is recruited to DNA repair foci in response to replication stalling. RNF4 depletion causes the accumulation of increased numbers of HU-induced BLM foci, whereas the numbers of RPA, RAD51, and gamma-H2AX foci are unaffected by fork stalling in RNF4-depleted cells. Sister chromatid exchanges are normal in RNF4-depleted cells. These data indicate that RNF4 can regulate BLM retention at stalled forks without affecting repair by homologous recombination. RNF4 depletion reduces cell proliferation and survival of untreated cells, and it confers hypersensitivity to replication stalling by HU. Studies of cell-cycle progression using bromodeoxyuridine incorporation and flow cytometry show that RNF4 depletion causes a delay of re-entry into S phase in HU-treated cells and the replication delay is partially rescued by BLM mutation. The delay is not caused by modification of checkpoint kinase activities. Analysis of replication dynamics using the DNA fiber assay show that RNF4 depletion causes an increase of permanently stalled replication forks and a decrease in activation of dormant origins following recovery from HU-induced replication stress. Co-depletion of RNF4 and BLM partially rescues these defects. Replication dynamics is unaffected by RNF4 depletion in untreated cells. These data indicate that RNF4 regulates BLM's functions in replication fork stability and dormant origin firing, without affecting BLM's roles in homologous recombination. We propose that sumoylation of BLM at stalled replication forks leads to RNF4-mediated ubiquitylation and subsequent proteasome-dependent degradation of BLM, which relieves BLM's active inhibition of dormant origin firing. Citation Format: Nathan A. Ellis, Wei-Chih Yang, Mary Yagle, Jianmei Zhu, Jing Huang, Michael Seidman, Michael J. Matunis. The SUMO-targeted ubiquitin ligase RNF4 regulates BLM helicase's function in dormant origin firing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3036. doi:10.1158/1538-7445.AM2015-3036