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

描述(申请人提供)：人类乳头瘤病毒(HPV)是乳头状瘤(疣)的病原体，几乎所有的宫颈癌和大多数肛门生殖器癌以及口腔/口咽癌都是由HPV引起的。除了是重要的人类病原体外，HPV还是真核细胞DNA复制的极佳模型。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)外，HPV完全依赖宿主蛋白来复制它们的基因组。初步数据表明，HPV复制蛋白与PolDNA之间存在直接的相互作用和功能关系，从而导致PolDNA合成活性增加。虽然在原核系统中已经证实了它，但在HPV复制系统中从未描述过解旋酶和前导链聚合酶之间的功能性偶联。这项拟议工作的范围是双重的；它将有助于阐明真核DNA复制分叉延长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基本相互作用，这些相互作用可能为药物干预提供潜在的靶点。因此，本项目的目的是研究人乳头瘤病毒复制蛋白(E1和E2)与细胞DNA聚合酶�(Poll�)之间的功能相互作用。通过使用不同的DNA模板，将分析在存在和不存在E1(和E2)和已知的聚合酶辅助因子的情况下，POL�的DNA合成活性，包括加工性、链延长效率、聚合酶回收和聚合酶负载量。相反，在存在和不存在聚合酶复合体的情况下，将分析E1的活性，以确定是否与POL�相互作用刺激E1功能。将通过检测解旋酶解离和解旋酶ATPase活性的功能分析来分析E1的活性。还将通过使用电泳迁移率改变分析(EMSA)来评估E2在PolDNA存在的情况下与�结合的能力，以及它与PolDNA形成复合体的能力。

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).