Replication Protein A (RPA) Phosphorylation Prevents RPA Association with Replication Centers

Vitaly M Vassin; Marc S Wold; James A Borowiec

doi:10.1128/MCB.24.5.1930-1943.2004

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Replication Protein A (RPA) Phosphorylation Prevents RPA Association with Replication Centers

Journal article

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Replication Protein A (RPA) Phosphorylation Prevents RPA Association with Replication Centers

Vitaly M Vassin, Marc S Wold and James A Borowiec

Molecular and cellular biology, Vol.24(5), pp.1930-1943

03/2004

DOI: 10.1128/MCB.24.5.1930-1943.2004

PMCID: PMC350552

PMID: 14966274

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Abstract

Mammalian replication protein A (RPA) undergoes DNA damage-dependent phosphorylation at numerous sites on the N terminus of the RPA2 subunit. To understand the functional significance of RPA phosphorylation, we expressed RPA2 variants in which the phosphorylation sites were converted to aspartate (RPA2D) or alanine (RPA2A). Although RPA2D was incorporated into RPA heterotrimers and supported simian virus 40 DNA replication in vitro, the RPA2D mutant was selectively unable to associate with replication centers in vivo. In cells containing greatly reduced levels of endogenous RPA2, RPA2D again did not localize to replication sites, indicating that the defect in supporting chromosomal DNA replication is not due to competition with the wild-type protein. Use of phosphospecific antibodies demonstrated that endogenous hyperphosphorylated RPA behaves similarly to RPA2D. In contrast, under DNA damage or replication stress conditions, RPA2D, like RPA2A and wild-type RPA2, was competent to associate with DNA damage foci as determined by colocalization with γ-H2AX. We conclude that RPA2 phosphorylation prevents RPA association with replication centers in vivo and potentially serves as a marker for sites of DNA damage.

DNA Dynamics and Chromosome Structure

Details

Title: Subtitle: Replication Protein A (RPA) Phosphorylation Prevents RPA Association with Replication Centers
Creators: Vitaly M Vassin - Department of Biochemistry and New York University Cancer Institute, New York University School of Medicine, New York, New York 10016
Marc S Wold - Department of Biochemistry and New York University Cancer Institute, New York University School of Medicine, New York, New York 10016
James A Borowiec - Department of Biochemistry and New York University Cancer Institute, New York University School of Medicine, New York, New York 10016
Resource Type: Journal article
Publication Details: Molecular and cellular biology, Vol.24(5), pp.1930-1943
DOI: 10.1128/MCB.24.5.1930-1943.2004
PMID: 14966274
PMCID: PMC350552
NLM abbreviation: Mol Cell Biol
ISSN: 0270-7306
eISSN: 1098-5549
Publisher: American Society for Microbiology
Language: English
Date published: 03/2004
Academic Unit: Radiation Oncology; Biochemistry and Molecular Biology
Record Identifier: 9984025278302771

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