biostudies-literatureRoshan PU.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)U.S. Department of Health & Human Services | NIH | NIH Office of the Director (OD)92https://www.ebi.ac.uk/biostudies/studies/S-EPMC12770403biostudies-literatureUnknown17(1)Replication protein A (RPA) is a heterotrimeric single-strand DNA binding protein essential for DNA metabolism. Segregation of RPA functions in response to DNA damage is fine-tuned by hyperphosphorylation of the RPA32 subunit that is dependent on cyclin-dependent kinase (CDK)-mediated priming phosphorylation at the Ser-23 and Ser-29 sites. However, the mechanism of priming-driven hyperphosphorylation of RPA and the modulation of cell cycle progression by the RPA-CDK axis remains unresolved. Here, we uncover that the RPA70 subunit is also phosphorylated by CDK1 at Thr-191. This modification is crucial for G2 to M phase transition. This function is enacted through reciprocal regulation of CDK1 activity via a feedback circuit espoused by stabilization of WEE1 kinase. The Thr-191 phosphosite oNature communicationsMechanism of RPA phosphocode priming and tuning by CDK1/WEE1 signaling circuit.PMC12770403R35GM149320S10OD030343R01GM143179Vayyeti ALuebbers RKaushik VAntony ERoshan PMistry ADeveryshetty JOriganti SAntony AfalseMechanism of RPA phosphocode priming and tuning by CDK1/WEE1 signaling circuit.Replication protein A (RPA) is a heterotrimeric single-strand DNA binding protein essential for DNA metabolism. Segregation of RPA functions in response to DNA damage is fine-tuned by hyperphosphorylation of the RPA32 subunit that is dependent on cyclin-dependent kinase (CDK)-mediated priming phosphorylation at the Ser-23 and Ser-29 sites. However, the mechanism of priming-driven hyperphosphorylation of RPA and the modulation of cell cycle progression by the RPA-CDK axis remains unresolved. Here, we uncover that the RPA70 subunit is also phosphorylated by CDK1 at Thr-191. This modification is crucial for G2 to M phase transition. This function is enacted through reciprocal regulation of CDK1 activity via a feedback circuit espoused by stabilization of WEE1 kinase. The Thr-191 phosphosite o2025-01-01T00:00:00Z2025 Dec2026-06-06T10:55:25.245Z2026-05-29T03:12:48.876ZS-EPMC127704034136588310.1038/s41467-025-66794-6