Simian virus 40 large T antigen binds to topoisomerase I.
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.
Simmons
D T
DT
Department of Biological Sciences, University of Delaware, Newark 19716-2590, USA. dsimmons@brahms.udel.edu
Melendy
T
T
Usher
D
D
Stillman
B
B
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D019915:DNA Primase; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D017354:Point Mutation; D011485:Protein Binding; D012316:RNA Nucleotidyltransferases / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013539:Simian virus 40 / Q000378:metabolism*; D018411:Spodoptera / Q000166:cytology
D000911:Antibodies, Monoclonal; D000952:Antigens, Polyomavirus Transforming; D011993:Recombinant Fusion Proteins; D004247:DNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004264:DNA Topoisomerases, Type I