Identification of Eukaryotic DNA Replication Proteins Using Simian Virus 40 in Vitro Replication System

George S. Brush, Thomas J. Kelly, Bruce Stillman

Research output: Contribution to journalArticlepeer-review

Abstract

At present, there is no in vitro DNA replication assay suitable for the direct analysis of chromosomal DNA replication in eukaryotes. Except in simple organisms such as yeast, the identification of replication origins and the proteins that bind to these sites has been extremely difficult. Eukaryotic DNA replication is tightly controlled during the cell cycle. It depends on the action of many regulatory proteins and to those directly involved in synthesizing DNA. One approach to circumvent these difficulties is to investigate the replication of viral DNA within eukaryotic cells. Viruses have small, well-defined genomes and the replication of viral DNA is free of some of the normal cellular constraints. Simian virus 40 (SV40) DNA replication requires only one viral protein; the large T antigen, which is an extremely useful tool for studying cellular DNA replication. The development of an in vitro SV40 DNA replication system I has led to the identification and characterization of many human replication proteins and has allowed for the isolation of homologous proteins from other eukaryotic species. This work has led to the realization that the basic replication machinery is conserved in all eukaryotes from yeast to man. SV40 DNA replication takes place within the nucleus of a permissive primate cell, where T antigen binds to and locally unwinds the circular SV40 minichromosome at the origin. This allows the host replication proteins to initiate bidirectional DNA synthesis and complete the duplication of the entire DNA molecule in a semidiscontinuous manner. SV40 DNA replication in vitro closely resembles the in vivo reaction and is dependent on the viral origin of replication, T antigen, and primate cell cytoplasmic extract. The cellular components necessary and sufficient for the replication of plasmid DNA containing the SV40 origin have been identified through fractionation of the cytoplasmic extract and reconstitution of activity.

Original languageEnglish (US)
Pages (from-to)522-548
Number of pages27
JournalMethods in Enzymology
Volume262
Issue numberC
DOIs
StatePublished - Jan 1 1995
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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