Abstracts Biological Sciences

Interactions between polyomavirus large T antigen and the viral replication origin DNA : how and why

by Yu-Cai Peng

Polyomavirus large T antigen is the major regulatory protein in the polyomavirus life cycle. It binds to multiple G(A/G)GGC pentanucleotide sequences in sites 1/2, A, B, and C within and adjacent to the origin of viral DNA replication on the polyomavirus genome. The nature of interactions between large T antigen and the viral origin of DNA replication is not fully understood. We set out to produce large T antigen protein in the methylotropic yeast Pichia pastoris by placing the large T antigen gene downstream of the strong alcohol oxidase (AOX1) promoter. Large T antigen was purified by immuno-affinity chromatography by using a monoclonal antibody. While optimizing the conditions for binding of large T antigen to viral origin DNA, we discovered that binding was substantially stronger at pH 6 to 7 than at pH 7.4 to 7.8, a range often used in DNA binding assays. We showed that increased binding at low pH is due to increased stability of protein-DNA complexes, and that large T antigen molecules self-associated at low pH, forming massive complexes. ATP increased binding of large T antigen to origin DNA by about 2-fold at pH 7.8, but had no detectable effect at pH 7 or below. Enhanced, stable DNA binding by large T antigen to viral origin DNA at pH 6 enabled us to develop a novel gel mobility shift assay using unfixed protein-DNA complexes. We demonstrated that this assay is very sensitive and highly specific. This method can be used both for detection of large T antigen in crude cell lysates and for quantitation of binding of purified large T antigen to target DNAs under various conditions. Using a series of point and deletion mutants in the viral origin of DNA replication, we demonstrated that binding of large T antigen to sites 1/2, A, B, and C is cooperative. Binding of large T antigen to one site stimulated binding to other sites 20 to 100 bp distant, and binding to inherently weak sites was strengthened if two or more such sites were present on the same DNA molecule. These findings suggest that large T antigen molecules bound to DNA interact with each other to mutually stabilise their binding. ATP was shown to stabilise large T antigen-DNA complexes against dissociation only if the DNA contained site 1/2. ATP specifically enhanced protection against DNase I digestion in the central 10 to 12 bp of site 1/2, where hexamers are believed to form and begin unwinding DNA. We propose a model in which large T antigen molecules bound to sites 1/2, A, B, and C on origin DNA form a compact protein-DNA complex via mutual interactions; large T antigen molecules bound to sites A, B, and C are mobilised and "handed over" to site 1/2, where ATP stimulates their assembly into hexamers.