Replication protein A mutants lacking phosphorylation sites for p34cdc2 kinase support DNA replication

L A Henricksen; M S Wold

doi:10.1016/S0021-9258(19)51069-9

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Replication protein A mutants lacking phosphorylation sites for p34cdc2 kinase support DNA replication

Journal article

Open access

Peer reviewed

Replication protein A mutants lacking phosphorylation sites for p34cdc2 kinase support DNA replication

L A Henricksen and M S Wold

The Journal of biological chemistry, Vol.269(39), pp.24203-24208

09/30/1994

DOI: 10.1016/S0021-9258(19)51069-9

PMID: 7929076

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Abstract

Replication Protein A (RPA) is a multisubunit, single-stranded DNA-binding protein essential for DNA metabolism in eukaryotic cells. The 32-kDa subunit of RPA is phosphorylated in a cell cycle-dependent manner becoming phosphorylated during S phase. It has been postulated that this phosphorylation may regulate the activities of RPA and that the family of p34cdc2 kinases directly catalyzes the phosphorylation of RPA in the cell. We have mutated the two consensus p34cdc2 sites in the 32-kDa subunit of RPA individually and in combination and purified the mutant protein complexes. Mutant RPA with both consensus p34cdc2 sites converted to alanine was not phosphorylated by purified p34cdc2 kinase. Nevertheless, we found that the properties of these RPA mutants were identical to those of the wild-type protein. The mutated RPA proteins had normal single-stranded DNA binding activity and were completely functional for DNA replication. In addition, the mutants became hyperphosphorylated when incubated under DNA replication conditions. These results demonstrate that phosphorylation by p34cdc2 kinase is not essential for RPA function in DNA replication in vitro. Possible roles of RPA phosphorylation on DNA metabolism are discussed.

Phosphorylation

DNA Replication

Mutation

CDC2 Protein Kinase - metabolism

DNA-Binding Proteins - metabolism

DNA, Viral - biosynthesis

Cloning, Molecular

Catalysis

DNA-Binding Proteins - genetics

Replication Protein A

Simian virus 40 - genetics

Simian virus 40 - metabolism

Details

Title: Subtitle: Replication protein A mutants lacking phosphorylation sites for p34cdc2 kinase support DNA replication
Creators: L A Henricksen - Department of Biochemistry, University of Iowa College of Medicine, Iowa City 52242
M S Wold
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.269(39), pp.24203-24208
DOI: 10.1016/S0021-9258(19)51069-9
PMID: 7929076
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: United States
Grant note: GM44721 / NIGMS NIH HHS
Language: English
Date published: 09/30/1994
Academic Unit: Radiation Oncology; Biochemistry and Molecular Biology
Record Identifier: 9984025253002771

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