The RING Finger E3 Ligase RNF25 Protects DNA Replication Forks Independently of its Canonical Roles in Ubiquitin Signaling

Lilly F. Chiou; Gaith N. Droby; Deepika Jayaprakash; Jay Ramanlal Anand; Xingyuan Zhang; Yang Yang; Christine A. Mills; Thomas S. Webb; Natalie K. Barker; Jialiu Xie; Di Wu; Laura E. Herring; Junya Tomida; Jessica L. Bowser; Cyrus Vaziri

doi:10.1038/s41467-025-62368-8

Molecular BiologyNature CommunicationsAug 5, 2025

Peer-reviewed journal article

The RING Finger E3 Ligase RNF25 Protects DNA Replication Forks Independently of its Canonical Roles in Ubiquitin Signaling

Lilly F. Chiou iD, Gaith N. Droby iD, Deepika Jayaprakash, Jay Ramanlal Anand iD, Xingyuan Zhang iD, Yang Yang iD, Christine A. Mills iD, Thomas S. Webb, Natalie K. Barker iD, Jialiu Xie iD, Di Wu iD, Laura E. Herring iD, Junya Tomida iD, Jessica L. Bowser iD, Cyrus Vaziri iD

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Abstract

The DNA damage response (DDR) mechanisms that allow cells to tolerate DNA replication stress are critically important for genome stability and cell viability. Using an unbiased genetic screen we identify a role for the RING finger E3 ubiquitin ligase RNF25 in promoting DNA replication stress tolerance. In response to DNA replication stress, RNF25-deficient cells generate aberrantly high levels of single-stranded DNA (ssDNA), accumulate in S-phase and show reduced mitotic entry. Using single-molecule DNA fiber analysis, we show that RNF25 protects reversed DNA replication forks generated by the fork remodeler HLTF from nucleolytic degradation by MRE11 and CtIP. Mechanistically, RNF25 interacts with the replication fork protection factor REV7 and recruits REV7 to nascent DNA after replication stress. The role of RNF25 in protecting replication forks is fully separable from its canonical functions in ubiquitin conjugation. This work reveals the RNF25-REV7 signaling axis as an important protective mechanism in cells experiencing replication stress.