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PMID: 14960720 Published · epublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Assembly of the replication initiation complex on SV40 origin DNA.

Nucleic acids research ·Vol. 32 ·No. 3 ·2004-00-00 ·Pages 1103-12

Simmons DT, Gai D, Parsons R, Debes A, Roy R

Abstract

The assembly of the complex that forms over the simian virus 40 origin to initiate DNA replication is not well understood. This complex is composed of the virus-coded T antigen and three cellular proteins, replication protein A (RPA), DNA polymerase alpha/primase (pol/prim) and topoisomerase I (topo I) in association with the origin. The order in which these various proteins bind to the DNA was investigated by performing binding assays using biotinylated origin DNA. We demonstrate that in the presence of all four proteins, pol/prim was essential to stabilize the initiation complex from the disruptive effects of topo I. At the optimal concentration of pol/prim, topo I and RPA bound efficiently to the complex, although pol/prim itself was not detected in significant amounts. At higher concentrations less topo I was recruited, suggesting that DNA polymerase is an important modulator of the binding of topo I. Topo I, in turn, appeared to be involved in recruiting RPA. RPA, in contrast, seemed to have little or no effect on the recruitment of the other proteins to the origin. These and other data suggested that pol/prim is the first cellular protein to interact with the double-hexameric T antigen bound to the origin. This is likely followed by topo I and then RPA, or perhaps by a complex of topo I and RPA. Stoichiometric analysis of the topo I and T antigen present in the complex suggested that two molecules of topo I are recruited per double hexamer. Finally, we demonstrate that DNA has a role in recruiting topo I to the origin.

MeSH Terms
Antigens, Polyomavirus Transforming/metabolism Base Sequence DNA Topoisomerases, Type I/metabolism DNA, Viral/chemistry,metabolism DNA-Binding Proteins/metabolism DNA-Directed DNA Polymerase/metabolism Macromolecular Substances Molecular Sequence Data Replication Origin Replication Protein A Simian virus 40/enzymology,genetics,physiology Virus Replication
Chemicals
Antigens, Polyomavirus Transforming DNA, Viral DNA-Binding Proteins Macromolecular Substances Replication Protein A DNA-Directed DNA Polymerase DNA Topoisomerases, Type I
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Simmons Daniel T
Department of Biological Sciences, University of Delaware, Newark, DE 19716-2590, USA. dsimmons@udel.edu
Gai Dahai
Parsons Rebekah
Debes Amanda
Roy Rupa
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
1362-4962
DOI
10.1093/nar/gkh236
Email
dsimmons@udel.edu
Published
2004-00-00
Epub
2004-00-11
Pages
1103-12
Language
English
Region
England
NLM ID
0411011
PMCID
PMC373383
Subset
IM
Grants
NCI NIH HHS · R01 CA036118 · United States
NCI NIH HHS · CA36118 · United States
External Links
PubMed source DOI: 10.1093/nar/gkh236 PMC Full Text View journal details
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