Initiation of simian virus 40 DNA replication in vitro: Large-tumor-antigen- and origin-dependent unwinding of the template

M. S. Wold, J. J. Li, T. J. Kelly

Research output: Contribution to journalArticlepeer-review

Abstract

Analysis of the kinetics of simian virus 40 (SV40) DNA replication in vitro demonstrated the existence of a slow presynthesis reaction that occurs prior to onset of extensive chain elongation and is dependent on a subset of the cellular proteins required for the complete replication reaction. When the presynthesis reaction is carried out in the presence of topoisomerase I, it is possible to detect extensive unwinding of the template DNA. This unwinding reaction is specific for templates that contain the wild-type SV40 origin of DNA replication and requires SV40 large tumor antigen (T antigen), ATP, and a protein fraction derived from HeLa cells. The required cellular protein may be a eukaryotic single-stranded-DNA-binding protein (SSB), since unwinding of the template is also observed when Escherichia coli SSB is substituted for the HeLa protein fraction. These observations suggest that during the initial stages of SV40 DNA replication, T antigen binds specifically to the viral origin and locally unwinds the DNA. This origin-dependent unwinding reaction is presumably a prerequisite for subsequent priming and elongation steps.

Original languageEnglish (US)
Pages (from-to)3643-3647
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume84
Issue number11
DOIs
StatePublished - 1987

ASJC Scopus subject areas

  • General

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