Simian virus 40 and polyomavirus large tumor antigens have different requirements for high-affinity sequence-specific DNA binding

Abstract

By using a DNA fragment immunoassay, the binding of SV40 and polyomavirus (Py) large tumor (T) antigens to regulatory regions at both viral origins of replication was examined. Although both Py T antigen and SV40 T antigen bind to multiple discrete regions on their proper origins and the reciprocal origin, several striking differences were observed. Py T antigen bound efficiently to 3 regions on Py DNA centered around an MboII site at nucleotide 45 (region A), a BglI site at nucleotide 92 (region B) and another MboII site at nucleotide 132 (region C). Region A is adjacent to the viral replication origin, and region C coincides with the major early mRNA cap site. Weak binding by Py T antigen to the origin palindrome centered at nucleotide 3 also was observed. SV40 T antigen binds strongly to Py regions A and B but only weakly to region C. This weak binding on region C was surprising because this region contains 4 tandem repeats of GPuGGC, the canonical pentanucleotide sequence thought to be involved in specific binding by T antigens. On SV40 DNA SV40 T antigen displayed its characteristic hierarchy of affinities, binding most efficiently to site 1 and less efficiently to site 2. Binding to site 3 was undetectable under these conditions. Py T antigen, despite an overall relative reduction of affinity for SV40 DNA, binds equally to fragments containing each of the 3 SV40 binding sites. Py T antigen, but not SV40 T antigen, also bound specifically to a region of human Alu DNA which bears a remarkabe homology to SV40 site 1. Both tumor antigens fail to precipitate DNA from the same region which has 2 direct repeats of GAGGC. These results indicate that despite similarities in protein structure and DNA sequence, requirements of the 2 T antigens for pentanucleotide configuration and neighboring sequence environment are different.