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PMID: 9811712 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

The replication protein A binding site in simian virus 40 (SV40) T antigen and its role in the initial steps of SV40 DNA replication.

Journal of virology ·Vol. 72 ·No. 12 ·1998-12-00 ·Pages 9771-81

Weisshart K, Taneja P, Fanning E

Abstract

Physical interactions of simian virus 40 (SV40) large tumor (T) antigen with cellular DNA polymerase alpha-primase (Pol/Prim) and replication protein A (RPA) appear to be responsible for multiple functional interactions among these proteins that are required for initiation of viral DNA replication at the origin, as well as during lagging-strand synthesis. In this study, we mapped an RPA binding site in T antigen (residues 164 to 249) that is embedded within the DNA binding domain of T antigen. Two monoclonal antibodies whose epitopes map within this region specifically interfered with RPA binding to T antigen but did not affect T-antigen binding to origin DNA or Pol/Prim, ATPase, or DNA helicase activity and had only a modest effect on origin DNA unwinding, suggesting that they could be used to test the functional importance of this RPA binding site in the initiation of viral DNA replication. To rule out a possible effect of these antibodies on origin DNA unwinding, we used a two-step initiation reaction in which an underwound template was first generated in the absence of primer synthesis. In the second step, primer synthesis was monitored with or without the antibodies. Alternatively, an underwound primed template was formed in the first step, and primer elongation was tested with or without antibodies in the second step. The results show that the antibodies specifically inhibited both primer synthesis and primer elongation, demonstrating that this RPA binding site in T antigen plays an essential role in both events.

MeSH Terms
Animals Antibodies, Monoclonal/pharmacology Antibodies, Viral/pharmacology Antigens, Polyomavirus Transforming/genetics,metabolism Binding Sites/genetics Cell Line DNA Polymerase I/metabolism DNA Primase/metabolism DNA Replication/physiology DNA-Binding Proteins/metabolism Epitope Mapping Humans Recombinant Fusion Proteins/genetics,metabolism Replication Protein A Simian virus 40/genetics,immunology,physiology Spodoptera
Chemicals
Antibodies, Monoclonal Antibodies, Viral Antigens, Polyomavirus Transforming DNA-Binding Proteins RPA1 protein, human Recombinant Fusion Proteins Replication Protein A DNA Primase DNA Polymerase I
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Weisshart K
Institute for Molecular Biotechnology, 07745 Jena, Germany.
Taneja P
Fanning E
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Article Info
Journal
Journal of virology
Abbr.
J Virol
ISSN
0022-538X
DOI
10.1128/JVI.72.12.9771-9781.1998
Published
1998-12-00
Pages
9771-81
Language
English
Region
United States
NLM ID
0113724
PMCID
PMC110488
Subset
IM
Grants
NIGMS NIH HHS · R01 GM052948 · United States
NIGMS NIH HHS · GM52948 · United States
External Links
PubMed source DOI: 10.1128/JVI.72.12.9771-9781.1998 PMC Full Text View journal details
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