Peng Y C, Acheson N H
Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
J Virol. 1998 Sep;72(9):7330-40. doi: 10.1128/JVI.72.9.7330-7340.1998.
Polyomavirus large T antigen binds to multiple 5'-G(A/G)GGC-3' pentanucleotide sequences in sites 1/2, A, B, and C within and adjacent to the origin of viral DNA replication on the polyomavirus genome. We asked whether the binding of large T antigen to one of these sites could influence binding to other sites. We discovered that binding to origin DNA is substantially stronger at pH 6 to 7 than at pH 7.4 to 7.8, a range often used in DNA binding assays. Large T antigen-DNA complexes formed at pH 6 to 7 were stable, but a fraction of these complexes dissociated at pH 7.6 and above upon dilution or during electrophoresis. Increased binding at low pH is therefore due at least in part to increased stability of protein-DNA complexes, and binding at higher pH values is reversible. Binding to fragments of origin DNA in which one or more sites were deleted or inactivated by point mutations was measured by nitrocellulose filter binding and DNase I footprinting. The results showed that large T antigen binds cooperatively to its four binding sites in viral DNA, suggesting that the binding of this protein to one of these sites stabilizes its binding to other sites via protein-protein contacts. Sites A, B, and C may therefore augment DNA replication by facilitating the binding of large T antigen to site 1/2 at the replication origin. ATP stabilized large T antigen-DNA complexes against dissociation in the presence, but not the absence, of site 1/2, and ATP specifically enhanced protection against DNase I digestion in the central 10 to 12 bp of site 1/2, at which hexamers are believed to form and begin unwinding DNA. We propose that large T antigen molecules bound to these multiple sites on origin DNA interact with each other to form a compact protein-DNA complex and, furthermore, that ATP stimulates their assembly into hexamers at site 1/2 by a "handover" mechanism mediated by these protein-protein contacts.
多瘤病毒大T抗原与多瘤病毒基因组上病毒DNA复制起点及其附近的1/2位点、A位点、B位点和C位点中的多个5'-G(A/G)GGC-3'五核苷酸序列结合。我们探究了大T抗原与这些位点之一的结合是否会影响其与其他位点的结合。我们发现，在pH 6至7时，大T抗原与起始DNA的结合比在pH 7.4至7.8时（DNA结合试验中常用的范围）要强得多。在pH 6至7形成的大T抗原-DNA复合物是稳定的，但在pH 7.6及以上时，这些复合物中的一部分在稀释或电泳过程中会解离。因此，低pH下结合增加至少部分是由于蛋白质-DNA复合物稳定性增加，而在较高pH值下的结合是可逆的。通过硝酸纤维素滤膜结合和DNase I足迹法测定了大T抗原与起始DNA片段的结合，这些片段中的一个或多个位点被点突变删除或失活。结果表明，大T抗原与其在病毒DNA中的四个结合位点协同结合，这表明该蛋白与这些位点之一的结合通过蛋白质-蛋白质相互作用稳定了其与其他位点的结合。因此，A位点、B位点和C位点可能通过促进大T抗原在复制起点与1/2位点的结合来增强DNA复制。ATP可稳定大T抗原-DNA复合物，防止其在存在1/2位点时解离，但在不存在1/2位点时则不然，并且ATP特异性增强了对1/2位点中央10至12 bp区域DNase I消化的保护作用，据信六聚体在该区域形成并开始解旋DNA。我们提出，结合在起始DNA上这些多个位点的大T抗原分子相互作用形成紧密的蛋白质-DNA复合物，此外，ATP通过由这些蛋白质-蛋白质相互作用介导的“交接”机制刺激它们在1/2位点组装成六聚体。
