Essential contact residues within SV40 large T antigen binding sites I and II identified by alkylation-interference

Abstract

Essential nucleotide contacts between the SV40 large T (tumor) antigen and binding sites I and II on the SV40 genome have been inferred from in vitro methylation- and ethylation-interference experiments. Each site contains two clusters of guanine residues that reduce the specific binding of T antigen when modified. Methylation at any one of nine guanines within site I or any one of five guanines within site II severely interferes with the interaction of T antigen with each respective site. Methylation at any one of a second group of five guanines within site II results in an appreciably weaker effect on the binding of T antigen. A similar inhibitory effect on binding is observed upon ethylation of adjacent phosphate residues. Although there are significant differences in the nucleotide sequence of the two binding sites, the pattern of protein contacts is strikingly similar between sites I and II. Three-dimensional projection reveals that the guanine contacts within each binding site are localized so that the specific binding interactions are accessible from only one face of the DNA helix.