PROTEIN-DNA INTERACTIONS IN PAPILLOMAVIRUS REPLICATION

了解DNA复制的生物化学及其相关的 真核生物细胞周期的调控在分子水平上是困难的 由于这些基因组的复杂性。作为替代方案，小的 染色体外基因组，如酵母ARS质粒和某些DNA 病毒，已被探索为细胞DNA复制的模型系统 以及它的规定。乳头瘤病毒是一个很有吸引力的模型系统 因为病毒基因组的复制是与 稳定感染期间的宿主细胞周期，这意味着病毒 基因组正在利用正常的细胞控制机制。长距离的 这项工作的目标将是识别和描述所有 细胞和病毒蛋白所需的真实的，受监管的 乳头瘤病毒复制。作为这一进程的第一步，立即 目标将是确定已知病毒蛋白的作用机制(S) 是体内复制所必需的。这项提案将把重点放在 特别是牛复制所需的两种病毒蛋白 人乳头瘤病毒(BPV)在体内，有E1和E2蛋白。最近的工作有 研究表明，E1蛋白与牛细小病毒DNA结合在一起的区域 体内复制的起源(威尔逊和鲁德斯-迈耶，手稿 提交)，并且该E1结合位点毗邻于 E2蛋白(V.Wilson，未发表的观察结果)。E1和E2都是 蛋白质将在细菌系统中表达并纯化，用于检测 在体外发挥作用。具体目标将是：1)确定地点， E1结合位点的体外组织和关键核苷酸，2) 确定E1结合对BPV DNA结构的影响，3)建立 复制活动是否需要源绑定 以及4)检查生理和功能之间的关系 E_1和E_2蛋白结合在起始区。这些研究应该 提供有关关键蛋白质-DNA相互作用的初步信息，网址为 BPV中复制的功能起始点。

Understanding the biochemistry of DNA replication and the associated regulation of cell cycle in eukaryotes is difficult at a molecular level due to the complexity of these genomes. As an alternative, small extrachromosomal genomes, such as yeast ARS plasmids and certain DNA viruses, have been explored as model systems for cellular DNA replication and its regulation. Papillomaviruses are an attractive model system because replication of the viral genome is coordinately regulated with the host cell cycle during stable infection, which implies that the viral genome is utilizing normal cellular control mechanisms. The long-range goal of this work will be the identification and characterization of all the cellular and viral proteins required for authentic, regulated papillomavirus replication. As a first step in this process, the immediate goal will be to define the mechanism(s) of action of viral proteins known to be required for replication in vivo. This proposal will focus specifically on the two viral proteins required for replication of bovine papillomavirus (BPV) in vivo, the E1 and E2 proteins. Recent work has shown that the E1 protein binds to BPV DNA in the region that functions as an origin of replication in vivo (Wilson and Ludes-Meyer, manuscript submitted), and that this E1 binding site is adjacent to a binding site for E2 protein (V. Wilson, unpublished observations). Both the E1 and E2 proteins will be expressed in a bacterial system and purified for assays of function in vitro. Specific aims will be to 1) define the location, organization, and critical nucleotides of the E1 binding site in vitro, 2) determine the effect of E1 binding on BPV DNA structure, 3) establish whether or not origin binding is required for the replication activity of E1 protein, and 4) examine the physical and functional relationship between the E1 and E2 proteins bound at the origin region. These studies should provide the initial information about critical protein-DNA interactions at a functional origin of replication in BPV.