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PMID: 8390661 Published · ppublish English Journal Article

Model system for DNA replication of a plasmid DNA containing the autonomously replicating sequence from Saccharomyces cerevisiae.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 90 ·No. 12 ·1993-06-15 ·Pages 5399-403

Ishimi Y, Matsumoto K

Abstract

A negatively supercoiled plasmid DNA containing autonomously replicating sequence (ARS) 1 from Saccharomyces cerevisiae was replicated with the proteins required for simian virus 40 DNA replication. The proteins included simian virus 40 large tumor antigen as a DNA helicase, DNA polymerase alpha.primase, and the multisubunit human single-stranded DNA-binding protein from HeLa cells; DNA gyrase from Escherichia coli, which relaxes positive but not negative supercoils, was included as a "swivelase." DNA replication started from the ARS region, proceeded bidirectionally with the synthesis of leading and lagging strands, and resulted in the synthesis of up to 10% of the input DNA in 1 h. The addition of HeLa DNA topoisomerase I, which relaxes both positive and negative supercoils, to this system inhibited DNA replication, suggesting that negative supercoiling of the template DNA is required for initiation. These results suggest that DNA replication starts from the ARS region where the DNA duplex is unwound by torsional stress; this unwound region can be recognized by a DNA helicase with the assistance of the multisubunit human single-stranded DNA-binding protein.

Related Genes
TRPI
MeSH Terms
Base Sequence DNA Replication DNA Topoisomerases, Type II/isolation & purification,metabolism DNA, Bacterial/genetics,isolation & purification,metabolism DNA, Fungal/genetics,isolation & purification,metabolism Escherichia coli/enzymology,genetics Gene Deletion Kinetics Molecular Sequence Data Plasmids Restriction Mapping Saccharomyces cerevisiae/genetics Tetracycline Resistance/genetics
Chemicals
DNA, Bacterial DNA, Fungal DNA Topoisomerases, Type II
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Ishimi Y
Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
Matsumoto K
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40 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.90.12.5399
Published
1993-06-15
Pages
5399-403
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC46727
Subset
IM
External Links
PubMed source DOI: 10.1073/pnas.90.12.5399 PMC Full Text View journal details
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