Replication protein A mutants lacking phosphorylation sites for p34cdc2 kinase support DNA replication.
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.
Henricksen
L A
LA
Department of Biochemistry, University of Iowa College of Medicine, Iowa City 52242.
Wold
M S
MS
D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002384:Catalysis; D003001:Cloning, Molecular; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D051716:Replication Protein A; D013539:Simian virus 40 / Q000235:genetics / Q000378:metabolism
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D051716:Replication Protein A; D016203:CDC2 Protein Kinase