| Abstract: | Essential nucleotide contacts between the polyomavirus large T antigen and its multiple specific binding regions within the regulatory sequences of the polyomavirus genome were determined in vitro by methylation interference. Methylation of any of the guanine residues of the 5'-G(A/G)GGC-3' pentanucleotide repeats in large-T-antigen-binding regions A, B, C, and 3 (A. Cowie and R. Kamen, J. Virol. 52:750-760, 1984) interfered with T antigen binding. Within regions A, B, and C these pentanucleotides are spaced 5 or 6 base pairs apart. Therefore, the clusters of contacted nucleotides within each of these binding regions are localized along one face of the DNA helix. Methylation of guanines within the sequences between the pentanucleotide repeats did not interfere with binding. The ORI binding region contains four additional pentanucleotide sequences within a region of dyad symmetry. Methylation of only particular guanines of these pentanucleotides interfered with T antigen binding. The spatial arrangement of the pentanucleotides in the ORI is such that the clusters of contacted guanines are situated around the DNA helix, thereby forming a very different arrangement from that found in the other binding regions. A model is discussed in which cooperative interactions between T antigen protomers, recognizing individual pentanucleotides, determines the strength and the function of different T antigen-DNA interactions. |
