Mechanism 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 on RPA70 is also crucial for priming hyperphosphorylation of RPA32 in response to DNA damage. Structurally, phosphorylation by CDK1 primes RPA by reconfiguring the domains to release the N-terminus of RPA32 and the two protein-interaction domains. These configurational changes markedly enhance the efficiency of multisite phosphorylation by other kinases independent of RPA-ssDNA interactions. Our findings establish a unique phosphocode-dependent feedback mechanism between RPA and RPA-regulating kinases that is fine-tuned to enact distinct bipartite functions in cell cycle progression and DNA damage response.