The role of the 34-kDa subunit of human replication protein A in simian virus 40 DNA replication in vitro

S. H. Lee, Kyoo Kim Dong Kyoo Kim

Research output: Contribution to journalArticle

82 Scopus citations

Abstract

Human replication protein A (RPA) is a three subunit protein complex involved in DNA replication, repair, and recombination. We investigated the role of the 34-kDa subunit (p34) of RPA in DNA replication by generating a series of p34 mutants. While deletion of the N-terminal domain of p34 prevented its phosphorylation by both cyclin-dependent kinase (Cdk) and DNA- dependent kinase, a double point mutant that lacks the major phosphorylation sites for Cdk could be phosphorylated by DNA-dependent kinase. In simian virus 40 (SV40) DNA replication, RPA containing either of these mutants functioned as efficiently as wild-type RPA. However, mutant RPA containing C- terminally deleted p34 was only marginally active. This indicates that the C- terminal region, but not the phosphorylation domain of p34, is necessary for RPA function in DNA replication. Furthermore, RPA containing the C-terminally deleted p34 mutant could stimulate DNA polymerase α, and bind to single- stranded DNAs but was limited in its ability to unwind DNA or interact with SV40 large T antigen (T Ag). These results suggest that RPA p34 interacts with SV40 TAg during the initiation of SV40 DNA replication and may be necessary for DNA unwinding.

Original languageEnglish (US)
Pages (from-to)12801-12807
Number of pages7
JournalJournal of Biological Chemistry
Volume270
Issue number21
DOIs
StatePublished - Jan 1 1995

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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