SIMIAN-VIRUS-40 LARGE T-ANTIGEN BINDS TO TOPOISOMERASE-I

Binding of simian virus 40 (SV40) large T antigen to human and calf th ymus topoisomerase I (topo I) was readily detected by using modified e nzyme-linked immunosorbent assays and immunoblots. In addition to WT T antigen, binding could also be readily demonstrated with T antigen fr agments from the amino-terminal region as well as with fragments missi ng this region, but much less so with small t antigen or with human p5 3. Antibody-blocking experiments showed that a monoclonal antibody tha t 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-bin ding regions in T antigen, one mapping within residues 82 to 246 and a n apparently weaker one present after residue 246. By comparing the bi nding 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 b inding sites for topo I and DNA polymerase overlap, whereas the one fo r RPA differs. Several unwinding-defective mutants of T antigen were p artially defective in their binding to topo I, suggesting that the bin ding to topo I is required for unwinding circular DNA. Finally, immuno precipitation experiments demonstrated that T antigen can interact wit h DNA-bound topo I, indicating that such an interaction may take place during SV40 DNA replication. (C) 1996 Academic Press, Inc.