Abstract 310: CHAF1A promotes the translesion DNA synthesis pathway in response to DNA replication stress

Abstract

Abstract Objective: Translesion DNA synthesis (TLS) pathway allows cancer cells to bypass the damage, allowing the blocked replication fork to move forward and helping cancer cells avoid genomic instability and cell death caused. Although some PCNA partners have previously been reported to participate in the TLS pathway, the mechanisms that regulate the TLS pathway remain unclear. Here, we revealed that CHAF1A is a new important regulator of TLS pathway. Methods: Cell models of CHAF1A gene knockdown (A549, KYSE510, KYSE450, etc.) were constructed by siRNA/shRNA knockdown method. We used DNA damage stimulation (HU, APH and UV) combined with western blotting to explore the effect of CHAF1A on PCNA monoubiquitination. The interaction of CHAF1A and its partners was demonstrated by immunoprecipitation and Duolink PLA assay. Cell fractionation assay was employed to measure protein binding to chromatin. DNA fiber assay was used to observe the condition of DNA replication fork. Micronucleus analysis was used to quantify cell micronucleus. The sensitivity of cells to DNA replication stress was tested by cloning formation assay and cell death assay. Kaplan-Meier survival analysis were used to analyze the clinical significance of CHAF1A expression. Results: CHAF1A enhances the interaction between PCNA and E3 ubiquitin protein ligase RAD18 and promotes PCNA monoubiquitination, thereby promoting the recruitment of Y-family DNA polymerase Pol η and enhancing cancer cell resistance to stimuli that trigger replication fork blockade. Mechanistically, CHAF1A-mediated PCNA monoubiquitination is independent of CHAF1A-PCNA interaction. CHAF1A interacts with both RAD18 and replication protein A2 (RPA2), mediating RAD18 binding on chromatin in response to DNA replication stress. Moreover, high expression of CHAF1A is significantly associated with poor prognosis in cancer patients. Conclusion: CHAF1A is a key TLS pathway regulator that is essential for cancer cell survival under DNA replication stress. The overall survival time of patients with high CHAF1A expression in lung, liver and esophageal cancers was significantly reduced. CHAF1A can be used as a key potential anti-tumor target, which has important clinical significance for enhancing the sensitivity of patients to radiotherapy and chemotherapy (This work was supported by the National Natural Science Foundation of China (82273108 and 82173034), the Innovative Team Grant of Guangdong Department of Education (2021KCXTD005), the Science and Technology Special Fund project of Guangdong Province in 2021 (No. 210729156901797) and the 2020 Li Ka Shing Foundation Cross-Disciplinary Research Grant (2020LKSFG07B)). Citation Format: Bing Wen, Li-Yan Xu, En-Min Li. CHAF1A promotes the translesion DNA synthesis pathway in response to DNA replication stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 310.