RNF185 orchestrates replication fork restart and homologous recombination through temporal RPA1 ubiquitination...

The replication protein A (RPA) complex safeguards single-stranded DNA (ssDNA) and coordinates repair during replication stress and homologous recombination (HR), but the mechanisms regulating its timely engagement and release at damage sites are not well defined. Here, we identified RNF185 as a critical E3 ligase that orchestrates temporally distinct patterns of RPA1 ubiquitination-shifting from K6/K63- to K48-linked chains-to precisely regulate HR and replication fork restart upon DNA damage. Mechanistically, RNF185 undergoes ATM/ATR-dependent phosphorylation at threonine 106 and translocates into the nucleus via interaction with NUP88 following DSBs. In the early response phase, RNF185 promotes K6/K63-linked ubiquitination of RPA1, stabilizing RPA1 on ssDNA to facilitate replication fork restart and efficient recruitment of RPA1 to damage sites. At later stages, RNF185 competes with the deubiquitinase OTUB1 for RPA1 binding and facilitates K48-linked ubiquitination at lysine 458, promoting RPA1 degradation and its removal from chromatin. Loss of RNF185 disrupts RPA1 turnover, impairs HR efficiency, destabilizes replication forks, and sensitizes tumor cells to irradiation and cisplatin. In vivo, RNF185 depletion significantly enhances the therapeutic efficacy of radiotherapy. In summary, this study demonstrates that RNF185 is a key regulatory factor in HR and replication fork restart, aiding cells in their response to radio- or chemotherapy induced DNA damage in clinical settings.