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PMID: 2174160 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Reconstitution of simian virus 40 DNA replication with purified proteins.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 87 ·No. 22 ·1990-11-00 ·Pages 8692-6

Weinberg DH, Collins KL, Simancek P, Russo A, Wold MS, Virshup DM, Kelly TJ

Abstract

Replication of plasmid DNA molecules containing the simian virus 40 (SV40) origin of DNA replication has been reconstituted with seven highly purified cellular proteins plus the SV40 large tumor (T) antigen. Initiation of DNA synthesis is absolutely dependent upon T antigen, replication protein A, and the DNA polymerase alpha-primase complex and is stimulated by the catalytic subunit of protein phosphatase 2A. Efficient elongation of nascent chains additionally requires proliferating cell nuclear antigen, replication factor C, DNA topoisomerase I, and DNA polymerase delta. Electron microscopic studies indicate that DNA replication begins at the viral origin and proceeds via intermediates containing two forks that move in opposite directions. These findings indicate that the reconstituted replication reaction has many of the characteristics expected of authentic viral DNA replication.

MeSH Terms
Antigens, Polyomavirus Transforming/physiology Cell-Free System DNA Replication In Vitro Techniques Microscopy, Electron Proteins/isolation & purification,physiology RNA, Viral/biosynthesis Simian virus 40/genetics
Chemicals
Antigens, Polyomavirus Transforming Proteins RNA, Viral
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
Weinberg D H
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Collins K L
Simancek P
Russo A
Wold M S
Virshup D M
Kelly T J
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34 references, click to expand
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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.87.22.8692
Published
1990-11-00
Pages
8692-6
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC55025
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.87.22.8692 PMC Full Text View journal details
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