PHI29 DNA POLYMERASE AND TERMINAL PROTEIN

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。DNA合成需要一个预先存在的启动子，通常是以寡核苷酸的形式。然而，几种病毒编码的DNA聚合酶，如肝炎和人腺病毒，独特地从末端蛋白开始复制，并从核苷酸开始延长。枯草杆菌的phi29噬菌体是最具生物化学特性的蛋白质启动复制系统，因此它可以作为这些更复杂的致病病毒的模型。尽管对Phi29 DNA复制的起始、过渡和延伸步骤进行了广泛的生化表征，但这些步骤都没有在原子水平上进行结构表征。因此，我们建议使用X射线结晶学来可视化Phi29DNA复制的三个步骤。我们已经确定了载脂蛋白聚合酶和与单链DNA络合的聚合酶的结构(手稿正在准备中)。这些结构为Phi29 DNA聚合酶显示的分子内链置换和加工提供了基础。我们目前正在确定起始复合体的结构(聚合酶及其启动蛋白和/或模板，以及传入的核苷酸)。我们还计划结晶并确定伸长和过渡络合物的结构。对这些新结构的分析将使我们能够理解phi29中的蛋白质启动。这样的理解将有助于洞察相关致病病毒的复制。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. DNA synthesis requires a preexisting primer for initiation, which is often in the form of an oligonucleotide. However, several viral-encoded DNA polymerases, such as hepatitis and human adenovirus, uniquely initiate replication from a ¿¿¿terminal protein¿¿¿ and prime elongation from a nucleotide. Phi29 bacteriophage of B. subtilis is the most biochemically well-characterized protein-primed replication system and it can therefore serve as a model for these more complex disease-causing viruses. Although there has been an extensive biochemical characterization of the initiation, transition, and elongation steps of Phi29 DNA replication, none of the steps have been structurally characterized at the atomic level. Therefore, we propose to use X-ray crystallography to visualize the three steps of Phi29DNA replication. We have already determined the structure of the apo polymerase and the polymerase complexed with single stranded DNA (manuscript in preparation). These structures have provided insight into the basis for the intramolecular strand displacement and processivity displayed by Phi29 DNA polymerase. We are currently determining the structure of the initiation complex (the polymerase with its priming protein, and/or template, and incoming nucleotide). We also plan to crystallize and determine the structures of the elongation and transition complexes. Analysis of these novel structures will allow us to understand protein priming in phi29. Such understanding will lend insight into the replication of related disease-causing viruses.