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bioRxiv · 10.1101/2025.01.16.633180

Mechanism of RPA phosphocode priming and tuning by Cdk1/Wee1 signaling circuit

Roshan, P.·Kaushik, V.·Mistry, A.·Vayyeti, A.·Antony, A.·Lubbers, R.·Deveryshetty, J.·Antony, E.·Origanti, S. S.
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Abstract

Replication protein A (RPA) is a heterotrimeric single-strand DNA binding protein that is integral to 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 remains unresolved. Furthermore, the modulation of cell cycle progression by the RPA-Cdk axis is not clearly understood. Here, we uncover that the RPA70 subunit is also phosphorylated by Cdk1 at Thr-191. This modification is crucial for the G2 to M phase transition. This function is enacted through reciprocal regulation of Cdk1 activity through 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 that markedly enhances the efficiency of multisite phosphorylation by other kinases. Our findings establish a unique phosphocode-dependent feedback mechanism between RPA and RPA-regulating kinases that is fine-tuned to enact bipartite functions in cell cycle progression and DNA damage response.

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Roshan, P., Kaushik, V., Mistry, A., Vayyeti, A., Antony, A., Lubbers, R., Deveryshetty, J., Antony, E., Origanti, S. S.. 2025-01-16. Mechanism of RPA phosphocode priming and tuning by Cdk1/Wee1 signaling circuit. https://doi.org/10.1101/2025.01.16.633180

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