Functional Interactions of HPV Replication Proteins E1 and E2 with Cellular DNA P

描述（由申请人提供）：人乳头瘤病毒（HPV）是乳头瘤（疣）的病原体，几乎是所有宫颈癌和大多数肛门生殖器癌以及口腔/口咽癌的原因。除了是重要的人类病原体之外，HPV还充当真核DNA复制的优良模型。除了PV复制蛋白E1和E2（分别是病毒复制解旋酶和转录调节蛋白）之外，HPV完全依赖宿主蛋白来复制其基因组。初步数据表明，HPV复制蛋白和pol β之间存在直接相互作用和功能关系，导致pol β的DNA合成活性增加。虽然在原核系统中已经证明了解旋酶和前导链聚合酶之间的功能性偶联，但在HPV复制系统中从未描述过。这项拟议工作的范围是双重的，它将有助于澄清真核DNA复制叉延伸背后的机制和相互作用，也将有助于确定病毒复制蛋白和宿主复制因子之间的重要相互作用，这些因子可能为药理学干预提供潜在靶点。因此，本项目的目的是表征HPV复制蛋白（E1和E2）和细胞DNA聚合酶（pol）之间的功能相互作用。通过使用各种DNA模板，将在存在和不存在E1（和E2）和已知聚合酶辅因子的情况下分析聚合酶的DNA合成活性，包括持续合成能力、链延伸效率、聚合酶循环和聚合酶加载。相反，将在存在和不存在聚合酶复合物的情况下分析E1的活性，以确定与聚合酶的相互作用是否刺激E1功能。将使用检查解旋酶解旋和解旋酶ATP酶活性的功能测定来分析E1活性。E2还将通过使用电泳迁移率变动分析（EMSA）评估其在存在聚合物的情况下结合DNA的能力，以及其与聚合物形成复合物的能力。

DESCRIPTION (provided by applicant): Human papillomaviruses (HPVs) are the causative agent of papillomas (warts) and are responsible for nearly all cervical and most anogenital and oral/oropharyngeal cancers. In addition to being important human pathogens, HPVs also serve as excellent models for eukaryotic DNA replication. Other than the PV replication proteins E1 and E2 (the viral replicative helicase and the transcriptional regulator protein, respectively), HPVs rely entirely on host proteins to replicate their genomes. Preliminary data demonstrates a direct interaction and a functional relationship between HPV replication proteins and pol �, which result in increased DNA synthesis activity of pol �. Though it has been demonstrated in prokaryotic systems, a functional coupling between helicase and leading strand polymerase has never been described in the HPV replication system. The scope of this proposed work is two-fold; it will help clarify the mechanisms and interactions behind eukaryotic DNA replication fork elongation and will also serve to identify essential interactions between viral replication protein and host replication factors that may provide potential targets for pharmacological intervention. Therefore, the aims of this project are to characterize the functional interactions between HPV replication proteins (E1 and E2) and cellular DNA polymerase � (pol �). Through the use of various DNA templates, the DNA synthesis activities of pol �, including processivity, chain elongation efficiency, polymerase recycling, and polymerase loading will be analyzed in the presence and absence of E1 (and E2) and known polymerase co-factors. Conversely, the activities of E1 will be analyzed in the presence and absence of the polymerase complex to determine if interaction with pol � stimulates E1 function. E1 activity will be analyzed using functional assays examining helicase unwinding and helicase ATPase activity. E2 will also be assessed for its ability to bind DNA in the presence of pol �, as well as its ability to form a complex with pol �, through the use of electrophoretic mobility shift assays (EMSAs).