The TopBP1-ATR axis is critical for maintaining genomic stability during DNA replication stress, yet the precise regulation of TopBP1 in replication stress responses remains poorly understood. In this study, we identified PHD and Ring Finger Domains 1 (PHRF1) as an important ATR activator through its interaction with TopBP1. Our analysis revealed a correlation between PHRF1 and genomic stability in cancer patients. Mechanistically, PHRF1 is recruited to DNA lesions in a manner dependent on its PHD domain and histone methylation. Subsequently, PHRF1 mono-ubiquitinates TopBP1 at lysine 73, which enhances the TopBP1-ATR interaction and activates ATR. Depletion of PHRF1 disrupts ATR activation and sensitizes cells to replication stress-inducing agents. Furthermore, conditional knockout of Phrf1 in mice leads to early lethality and impaired ATR-Chk1 axis signaling. Collectively, our findings establish PHRF1 as a novel E3 ligase for TopBP1, coordinating the replication stress response by enhancing TopBP1-ATR signaling.
© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.