Preferred DNA-binding-sites of polyomavirus large T-antigen.

Abstract

Polyomavirus large T-antigen is a multifunctional protein. Its essential function in virus infection is to control the synthesis of viral RNA and DNA. For this activity specific DNA binding is necessary. Large T-antigen can bind to several sites in the regulatory region of viral DNA, consisting of clustered GRGGC nucleotide motifs. Since large T-antigen also regulates the activity of cellular genes and cellular DNA synthesis, it seemed possible that there are alternative, as yet unrecognized, binding sites. To identify sites preferred by large T-antigen, double-stranded polynucleotides with random sequence were used. These polymers had a 31-bp central segment synthesized from a mixture of all four nucleotides and flanking segments of defined sequence. They were subjected to several cycles of binding to large T-antigen with intervening PCR amplification. Individual polynucleotides with affinity for large T-antigen were then isolated by cloning and their nucleotide sequences were determined. The majority of the polynucleotides contained two or three GRGGC motifs separated by between five and eight variable nucleotides. This result suggests that there are not any alternative high-affinity binding sites of large T-antigen. By comparing all the individual binding motifs an extended consensus sequence was observed. The dinucleotide TG was predominant immediately 5' to the binding pentanucleotide. On the 3'-side, at position +2, C residues were very rare. Although the pentanucleotide motif is the same as in polyomavirus DNA, the extended consensus sequence is not observed in viral DNA. In semi-quantitative experiments, binding of purified large T-antigen to a few of the selected DNA molecules was tested. Stable complexes were formed with DNA substrates containing two or three binding motifs in tandem. Binding to DNA with only one complete motif was weaker, but significantly stronger than non-specific association. This result has implications for the number of large T-antigen binding sites in cellular DNA. When mutant bc1081 large T-antigen, that is defective in specific DNA binding, was used in selection of polynucleotides, a different result was obtained. Neither bc1081 nor wild-type large T-antigen bound strongly to these polynucleotides. However, the presence of the motif TTCGGCTT, or part of it, in five of the six isolated polynucleotides suggested that the T-antigen selection was specific.