PXD068080

PXD068080 is an original dataset announced via ProteomeXchange.

Title	Mechanism of RPA phosphocode priming and tuning by Cdk1/Wee1 signaling circuit
Description	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 and the modulation of cell cycle progression by the RPA-Cdk axis remain 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 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. 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.
HostingRepository	PRIDE
AnnounceDate	2026-01-12
AnnouncementXML	Submission_2026-01-11_16:33:36.656.xml
DigitalObjectIdentifier	https://dx.doi.org/10.6019/PXD068080
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Monika Tokmina-Lukaszewska
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: NEWT:9606;
ModificationList	phosphorylated residue
Instrument	Orbitrap Eclipse

Revision	Datetime	Status	ChangeLog Entry
0	2025-09-04 09:45:04	ID requested
⏵ 1	2026-01-11 16:33:37	announced

10.6019/PXD068080;

10.1038/s41467-025-66794-6;

submitter keyword: RPA domain configurations,RPA phosphorylation, Cdk1, Wee1, cell cycle

Edwin Antony
contact affiliation	Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO, USA
contact email	edwin.antony@health.slu.edu
lab head
Monika Tokmina-Lukaszewska
contact affiliation	Montana State University
contact email	tokminalukas@montana.edu
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/01/PXD068080

PRIDE project URI

• PRIDE
  1. PXD068080
     1. Label: PRIDE project
     2. Name: Mechanism of RPA phosphocode priming and tuning by Cdk1/Wee1 signaling circuit