Mechanism of RPA phosphocode priming and tuning by CDK1/WEE1 signaling circuit - research article

Q: What specific role does CDK1 play in RPA regulation according to the article?

CDK1 phosphorylates the RPA70 subunit at Thr-191, which is required for proper G2-to-M transition and helps prime subsequent RPA32 hyperphosphorylation after DNA damage.

Q: Why are the Ser-23 and Ser-29 sites on RPA32 important?

These sites act as priming phosphorylation targets that enable hyperphosphorylation of RPA32 and help fine-tune RPA function in response to DNA damage and cell-cycle progression.

Q: How does phosphorylation by CDK1 change RPA to improve phosphorylation efficiency?

CDK1-mediated phosphorylation reconﬁgures RPA domains to release the N-terminus of RPA32 and two protein-interaction domains, markedly enhancing multisite phosphorylation by other kinases independent of RPA–ssDNA interactions.

Sep 29, 2026

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RPA (Replication Protein A) is a heterotrimeric single-strand DNA–binding protein whose functions are regulated through hyperphosphorylation of the RPA32 subunit. The mechanism linking CDK-mediated priming at Ser-23 and Ser-29 to RPA regulation of cell-cycle progression remains unresolved. This work identifies CDK1-dependent phosphorylation of the RPA70 subunit at Thr-191 and shows its importance for the G2-to-M transition. The Thr-191 phosphosite enables priming hyperphosphorylation of RPA32 after DNA damage via reciprocal CDK1–WEE1 feedback and domain reconﬁguration that enhances multisite phosphorylation efficiency.

LanguageEnglish

CategoryResearch & Report

FormatPDF

Pages19

Reading Time48 min

AccessFree to download

Abstract

RPA regulation by hyperphosphorylation

CDK1 phosphorylation of RPA70 (Thr-191)

Reciprocal CDK1–WEE1 feedback mechanism

Structural basis for priming and multisite phosphorylation

Article [https://doi.org/10.1038/s41467-025-66794-6](https://doi.org/10.1038/s41467-025-66794-6) Mechanism of RPA phosphocode priming and tuning by CDK1/WEE1 signaling circuit Received: 24 March 2025 | Accepted: 14 November 2025 | | --- | | | | Check for updates | Poonam Roshan1,4, Vikas Kaushik2,4, Ayush Mistry 2, Abhinav Vayyeti2, Aidan Antony2,3, Riley Luebbers1, Jaigeeth Deveryshetty 2, Edwin Antony 2 & Soﬁa Origanti1 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 ofthe RPA32 subunit that is dependent on cyclin-dependent kinase (CDK)-mediated priming phosphorylation atthe Ser-23 and Ser-29 sites. However, the mechanism of priming-driven hyperphosphorylation ofRPA 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. Replication Protein A (RPA) is a single-strand DNA (ssDNA) binding protein that functions in nearly all aspects of DNA metabolism, including repair, replication, and recombination1,2. To support these diverse activities, RPA utilizes a modular and ﬂexible architecture in which multiple oligonucleotide/oligosaccharide-binding (OB) domains are connected by disordered linkers1,3–6. Each OB-domain contributes to deﬁned DNA-binding or protein-interaction functions2,7. RPA is a heterotrimer composed of RPA70 (RFA1), RPA32 (RFA2), and RPA14 (RFA3) subunits that are structurally conﬁgured as six OB-domains (Fig. 1a). The OB-domains are functionally classiﬁed as DNA-binding domains (DBDs) or protein-interaction domains (PIDs) . DBDs-A, B, and C reside in RPA70 and DBD-D in RPA32, respectively. PID70N is situated at the N-terminus of RPA70 and tethered to DBD-A through a long-disordered F-A linker. Similarly, PID32C (also commonly termed as the winged-helix (wh) motif) is situated at the C-terminus of RPA32 and connected to DBD-D by a disordered D-wh linker. RPA binds to ssDNA with high afﬁnity (KD < 1 nM) and all four DBDs contribute to this property1,2. DBDs-A and B are more dynamic compared to DBDs-C and D7–10. Domains C, D, and E interact to form the trimerization core (TriC) that holds the RPA complex together3. The versatility of RPA in DNA metabolism arises from conﬁgurational plasticity among its domains, where different domain and linker arrangements generate conﬁgurations suited to speciﬁc functional contexts2,5,11,12. 1Department of Biology, Saint Louis University, St. Louis, MO, USA. 2Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO, USA. 3Ladue Horton Watkins High School, Saint Louis, MO, USA. 4These authors contributed equally: Poonam Roshan, Vikas Kaushik. e-mail: [edwin.antony@health.slu.edu](edwin.antony@health.slu.edu); soﬁ[a.origanti@slu.edu](a.origanti@slu.edu) We recently demonstrated that such conﬁgurational transitions are further modulated by phosphorylation9,10. Most known phosphosites in RPA cluster within a ~ 40 aa disordered regio

FAQs

What specific role does CDK1 play in RPA regulation according to the article?

CDK1 phosphorylates the RPA70 subunit at Thr-191, which is required for proper G2-to-M transition and helps prime subsequent RPA32 hyperphosphorylation after DNA damage.

Why are the Ser-23 and Ser-29 sites on RPA32 important?

These sites act as priming phosphorylation targets that enable hyperphosphorylation of RPA32 and help fine-tune RPA function in response to DNA damage and cell-cycle progression.

How does phosphorylation by CDK1 change RPA to improve phosphorylation efficiency?

CDK1-mediated phosphorylation reconﬁgures RPA domains to release the N-terminus of RPA32 and two protein-interaction domains, markedly enhancing multisite phosphorylation by other kinases independent of RPA–ssDNA interactions.

Sep 29, 2026

Upload by Mia