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PMID: 2157141 Published · ppublish English Journal Article

Simian virus 40 origin auxiliary sequences weakly facilitate T-antigen binding but strongly facilitate DNA unwinding.

Molecular and cellular biology ·Vol. 10 ·No. 4 ·1990-04-00 ·Pages 1719-28

Gutierrez C, Guo ZS, Roberts J, DePamphilis ML

Abstract

The complete simian virus 40 (SV40) origin of DNA replication (ori) consists of a required core sequence flanked by two auxiliary sequences that together increase the rate of DNA replication in monkey cells about 25-fold. Using an extract of SV40-infected monkey cells that reproduced the effects of ori-auxiliary sequences on DNA replication, we examined the ability of ori-auxiliary sequences to facilitate binding of replication factors and to promote DNA unwinding. Although the replicationally active form of T antigen in these extracts had a strong affinity for ori-core, it had only a weak but specific affinity for ori-auxiliary sequences. Deletion of ori-auxiliary sequences reduced the affinity of ori-core for active T antigen by only 1.6-fold, consistent with the fact that saturating concentrations of T antigen in the cell extract did not reduce the stimulatory role of ori-auxiliary sequences in replication. In contrast, deletion of ori-auxiliary sequences reduced the efficiency of ori-specific, T-antigen-dependent DNA unwinding in cell extracts at least 15-fold. With only purified T antigen in the presence of topoisomerase I to unwind purified DNA, ori-auxiliary sequences strongly facilitated T-antigen-dependent DNA conformational changes consistent with melting the first 50 base pairs. Under these conditions, ori-auxiliary sequences had little effect on the binding of T antigen to DNA. Therefore, a primary role of ori-auxiliary sequences in DNA replication is to facilitate T-antigen-dependent DNA unwinding after the T-antigen preinitiation complex is bound to ori-core.

MeSH Terms
Animals Antigens, Polyomavirus Transforming Binding, Competitive Cell Line Cloning, Molecular DNA Helicases DNA Replication DNA, Circular/genetics DNA, Viral/genetics,metabolism DNA-Binding Proteins/metabolism Kinetics Models, Genetic Nucleic Acid Conformation Plasmids Restriction Mapping Simian virus 40/genetics,immunology
Chemicals
Antigens, Polyomavirus Transforming DNA, Circular DNA, Viral DNA-Binding Proteins DNA Helicases
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Gutierrez C
Department of Cell and Developmental Biology, Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
Guo Z S
Roberts J
DePamphilis M L
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/mcb.10.4.1719-1728.1990
Published
1990-04-00
Pages
1719-28
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC362278
Subset
IM
External Links
PubMed source DOI: 10.1128/mcb.10.4.1719-1728.1990 PMC Full Text View journal details
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