Simmons, D. T., Melendy, T., Usher, D., Stillman, B. (August 1996) Simian virus 40 large T antigen binds to topoisomerase I. Virology, 222 (2). pp. 365-74. ISSN 0042-6822 (Print)
Abstract
Binding of simian virus 40 (SV40) large T antigen to human and calf thymus topoisomerase I (topo I) was readily detected by using modified enzyme-linked immunosorbent assays and immunoblots. In addition to WT T antigen, binding could also be readily demonstrated with T antigen fragments from the amino-terminal region as well as with fragments missing this region, but much less so with small t antigen or with human p53. Antibody-blocking experiments showed that a monoclonal antibody that binds to the N-terminal region and several antibodies that recognize the central region of T antigen interfere with the binding to topo I. Our data are consistent with the existence of two separate topo I-binding regions in T antigen, one mapping within residues 82 to 246 and an apparently weaker one present after residue 246. By comparing the binding of T antigen to topo I with that of T antigen to DNA polymerase alpha or RPA, a single-stranded DNA-binding protein, it was determined that the T antigen-topo I interaction is much stronger and that the binding sites for topo I and DNA polymerase overlap, whereas the one for RPA differs. Several unwinding-defective mutants of T antigen were partially defective in their binding to topo I, suggesting that the binding to topo I is required for unwinding circular DNA. Finally, immunoprecipitation experiments demonstrated that T antigen can interact with DNA-bound topo I, indicating that such an interaction may take place during SV40 DNA replication.
| Item Type: | Paper |
|---|---|
| Uncontrolled Keywords: | Animals Antibodies Monoclonal metabolism Antigens Polyomavirus Transforming genetics/*metabolism Cattle Cell Line DNA metabolism DNA Primase DNA Replication DNA Topoisomerases Type I genetics metabolism Humans Immunoblotting Point Mutation Protein Binding RNA Nucleotidyltransferases metabolism Rabbits Recombinant Fusion Proteins genetics metabolism Simian virus 40 metabolism Spodoptera cytology |
| Subjects: | organism description > virus |
| CSHL Authors: | |
| Communities: | CSHL labs > Stillman lab |
| Highlight: | Stillman, Bruce W. |
| Depositing User: | CSHL Librarian |
| Date: | 15 August 1996 |
| Date Deposited: | 02 Mar 2012 15:53 |
| Last Modified: | 20 Jun 2017 19:38 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/25055 |
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