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 alpha, 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 T Ag during the initiation of SV40 DNA replication and may be necessary for DNA unwinding.
Highlights
11 Supported by a fellowship from Inje Foundation, Kimhae, Korea. 1 The abbreviations used are: SV40, simian virus 40; pol a and S, DNA polymerase a and S, respectively; topo, topoisomerase; PCR, polymerase chain reaction; RPA, replication protein A; SSB, single stranded DNAbinding protein; ssDNA, single-stranded DNA;T Ag, SV40 large tumor antigen; DTT,dithiothreitol; TBE, Tris borate-EDTA buffer; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; cyclin-dependent kinase (Cdk), cyelin-dependent kinase; kb, kilobase pairfs); ABTS, 2,2-azinobis(3-ethyl-benzothiazoline-6-sulfonic acid
SV40 T Ag, RPA, and the DNA polymerase o-primase complex functionally interact in vitro to form a primosome complex that is required for both primer synthesis (Collins and Kelly, 1991; Melendy and Stillman, 1993) and DNA synthesis at the replication fork (Murakami and Hurwitz, 1993)
We address the role of RPA p34 in DNA replication by comparing the function of wild-type RPA with that ofa series of mutants
Summary
Vol 270, No 21, Issue of May 26, pp. 12801-12807, 1995 Printed in U.S.A. The Role of the 34-kDa Subunit of Human Replication Protein A in Simian Virus 40 DNA Replication in Vitro*. Mutant RPA containing C-terminally deleted p34 was only marginally active This indicates that the C-terminal region, but not the phosphorylation domain ofp, is necessary for RPA function in DNA replication. RPA containing the C-terminally deleted p34 mutant could stimulate DNA polymerase a, 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 T Ag during the initiation of SV40 DNA replication and may be necessary for DNA unwinding. Deletion of the N-terminal region of RPA p34 abolished its phosphorylation by both Cdk and DNAdependent kinase but did not affect the mutant's ability to support SV40 DNA replication in vitro. We discuss the implications of these results on the role ofRPA p34 in DNA replication
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