PREFERRED DNA-BINDING-SITES OF POLYOMAVIRUS LARGE T-ANTIGEN

Polyomavirus large T-antigen is a multifunctional protein. Its essenti al function in virus infection is to control the synthesis of viral RN A 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-an tigen also regulates the activity of cellular genes and cellular DNA s ynthesis, it seemed possible that there are alternative, as yet unreco gnized, 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 al l four nucleotides and flanking segments of defined sequence. They wer e subjected to several cycles of binding to large T-antigen with inter vening PCR amplification. Individual polynucleotides with affinity for large T-antigen were then isolated by cloning and their nucleotide se quences were determined. The majority of the polynucleotides contained two or three GRGGC motifs separated by between five and eight variabl e 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 pen tanucleotide. On the 3'-side, at position +2, C residues were very rar e. Although the pentanucleotide motif is the same as in polyomavirus D NA, the extended consensus sequence is not observed in viral DNA. In s emi-quantitative experiments, binding of purified large T-antigen to a few of the selected DNA molecules was tested. Stable complexes were f ormed with DNA substrates containing two or three binding motifs in ta ndem. Binding to DNA with only one complete motif was weaker, but sign ificantly stronger than non-specific association. This result has impl ications for the number of large T-antigen binding sites in cellular D NA. When mutant bc1081 large T-antigen, that is defective in specific DNA binding, was used in selection of polynucleotides, a different res ult was obtained. Neither bc1081 nor wild-type large T-antigen bound s trongly to these polynucleotides. However, the presence of the motif ( TTGCTT)-T-CG, or part of it, in five of the six isolated polynucleotid es suggested that the T-antigen selection was specific.