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PMID: 2542940 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Purification of replication protein C, a cellular protein involved in the initial stages of simian virus 40 DNA replication in vitro.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 86 ·No. 10 ·1989-05-00 ·Pages 3584-8

Virshup DM, Kelly TJ

Abstract

The replication of simian virus 40 (SV40) DNA is dependent upon a single viral protein [tumor (T) antigen] and multiple cellular proteins. To define the required cellular proteins, we have made use of a cell-free system that supports the replication of plasmid DNA molecules containing the SV40 origin of replication. We report here the purification from HeLa cell extracts of replication protein C (RP-C), a previously undescribed protein that is required to reconstitute efficient DNA replication in vitro. Highly purified preparations of RP-C contain two closely related polypeptides of 32 and 34 kDa. Preincubation of purified RP-C with T antigen and the DNA template largely eliminates the delay normally observed before the onset of rapid DNA synthesis. In addition, RP-C stimulates the unwinding of duplex DNA molecules containing the SV40 replication origin in a reaction that requires T antigen and a single-stranded DNA binding protein. These observations suggest that RP-C is involved in the initial steps of SV40 DNA replication in vitro.

MeSH Terms
Antigens, Polyomavirus Transforming/physiology Cell Fractionation DNA Helicases/isolation & purification,physiology DNA Replication HeLa Cells In Vitro Techniques Kinetics Molecular Weight Simian virus 40/genetics,growth & development Viral Proteins/isolation & purification,physiology Virus Replication
Chemicals
Antigens, Polyomavirus Transforming Viral Proteins DNA Helicases
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Virshup D M
Department of Molecular Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Kelly T J
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.86.10.3584
Published
1989-05-00
Pages
3584-8
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC287182
Subset
IM
Grants
NCI NIH HHS · CA-16519 · United States
NCI NIH HHS · CA-40414 · United States
NIDDK NIH HHS · DK-01528 · United States
External Links
PubMed source DOI: 10.1073/pnas.86.10.3584 PMC Full Text View journal details
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