An interaction between replication protein A and SV40 T antigen appears essential for primosome assembly during SV40 DNA replication

Melendy, T., Stillman, B. (1993) An interaction between replication protein A and SV40 T antigen appears essential for primosome assembly during SV40 DNA replication. J Biol Chem, 268 (5). pp. 3389-95. ISSN 0021-9258 (Print)

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URL: http://www.ncbi.nlm.nih.gov/pubmed/8381428

Abstract

Replication protein A from human cells (hRPA) is a multisubunit single-stranded DNA-binding protein (ssb) and is essential for SV40 DNA replication in vitro. The related RPA from Saccharomyces cerevisiae (scRPA) is unable to substitute for hRPA in SV40 DNA replication. To understand this species specificity, we evaluated human and yeast RPA in enzymatic assays with SV40 T antigen (TAg) and human DNA polymerase alpha/primase, the factors essential for initiation of SV40 DNA replication. Both human and yeast RPA stimulated the polymerase and (at subsaturating levels of RPA) the primase activities of human DNA polymerase alpha/primase on homopolymer DNA templates. In contrast, both human and yeast RPA inhibited synthesis by DNA polymerase alpha/primase on natural single-stranded DNA (ssDNA) templates. T antigen reversed the inhibition of DNA polymerase alpha/primase activity on hRPA-coated natural ssDNA, as previously described, but was unable to reverse the inhibition on scRPA or Escherichia coli ssb-coated templates. Therefore, the ability of an ssb to reconstitute SV40 DNA replication correlated with its ability to allow the TAg stimulation of polymerase alpha/primase in this assay. Enzyme-linked immunoassays demonstrated that hRPA interacts with TAg, as previously described; however, scRPA does not bind to TAg in this assay. These and other recent results suggest that T antigen contains a function analogous to some prokaryotic DNA replication proteins that facilitate primosome assembly on ssb-coated template DNAs.

Item Type: Paper
Uncontrolled Keywords: Animals Antigens Polyomavirus Transforming metabolism Baculoviridae genetics Cell Line Comparative Study DNA Primase DNA Replication DNA Single-Stranded genetics/metabolism DNA-Binding Proteins metabolism DNA-Directed DNA Polymerase metabolism Enzyme-Linked Immunosorbent Assay Humans Insects Kinetics Models Biological RNA Nucleotidyltransferases metabolism Replication Factor A Saccharomyces cerevisiaemetabolism Simian virus 40 genetics metabolism
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > DNA replication
organism description > virus
CSHL Authors:
Communities: CSHL labs > Stillman lab
Highlight: Stillman, Bruce W.
Depositing User: CSHL Librarian
Date Deposited: 02 Mar 2012 19:40
Last Modified: 20 Jun 2017 19:58
Related URLs:
URI: http://repository.cshl.edu/id/eprint/25020

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