T antigen and template requirements for SV40 DNA replication in vitro

Stillman, B., Gerard, R. D., Guggenheimer, R. A., Gluzman, Y. (1985) T antigen and template requirements for SV40 DNA replication in vitro. EMBO Journal, 4 (11). pp. 2933-2939. ISSN 0261-4189

URL: http://www.ncbi.nlm.nih.gov/pubmed/2998767

Abstract

A cell-free system for replication of SV40 DNA was used to assess the effect of mutations altering either the SV40 origin of DNA replication or the virus-encoded large tumor (T) antigen. Plasmid DNAs containing various portions of the SV40 genome that surround the origin of DNA replication support efficient DNA synthesis in vitro and in vivo. Deletion of DNA sequences adjacent to the binding sites for T antigen either reduce or prevent DNA synthesis. This analysis shows that sequences that had been previously defined by studies in vivo to constitute the minimal core origin sequences are also necessary for DNA synthesis in vitro. Five mutant T antigens containing amino acid substitutions that affect SV40 replication have been purified and their in vitro properties compared with the purified wild-type protein. One protein is completely defective in the ATPase activity of T antigen, but still binds to the origin sequences. Three altered proteins are defective in their ability to bind to origin DNA, but retain ATPase activity. Finally, one of the altered T antigens binds to origin sequences and contains ATPase activity and thus appears like wild-type for these functions. All five proteins fail to support SV40 DNA replication in vitro. Interestingly, in mixing experiments, all five proteins efficiently compete with the wild-type protein and reduce the amount of DNA replication. These data suggest that an additional function of T antigen other than origin binding or ATPase activity, is required for initiation of DNA replication.

Item Type: Paper
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > DNA replication
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification
bioinformatics > genomics and proteomics > genetics & nucleic acid processing
bioinformatics > genomics and proteomics
CSHL Authors:
Communities: CSHL labs > Stillman lab
Highlight: Stillman, Bruce W.
Depositing User: Matt Covey
Date Deposited: 15 Nov 2013 21:59
Last Modified: 20 Jun 2017 20:38
PMCID: PMC554601
URI: http://repository.cshl.edu/id/eprint/28867

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