FUNCTIONAL DOMAINS OF THE HSV-1 UL42 PROTEIN

单纯疱疹病毒（HSV）提供了一个易于处理的遗传模型系统， 真核DNA复制，因为它编码至少7种蛋白质， 直接参与病毒DNA的合成， 对于病毒复制至关重要。 其中两种蛋白质负责 用于延长DNA链--DNA聚合酶（pol）催化核心 及其辅助蛋白UL 42。 这些蛋白质形成稳定的复合物， 体外并一起进行进行DNA合成。 长期目标 该项目的目的是了解UL 42在DNA复制中的作用， 它是如何与聚合物相互作用进行DNA复制的。 是 假设干扰UL 42与 pol将阻断病毒DNA合成。 因此， 对于它们的相互作用至关重要的UL 42和pol的结合提供了靶点 用于开发新型抗病毒化合物。 由于结构 pol辅助蛋白在高度分化的物种中的保守性， 确定这个复合体的结构可能会导致 甚至更广泛的方法来合理设计化合物， 抑制肿瘤细胞的DNA合成。 的具体目标 项目是1）确定UL 42对DNA的作用机制 使用瞬时动力学分析聚合; 2）使用酵母双- 识别UL 42和轮询的子域的混合系统能够 体内物理相互作用; 3）选择非相互作用的pol和 酵母双杂交系统中的UL 42突变体和基因外抑制子 在使用PCR或寡核苷酸定向诱变进行随机诱变后， 改变带电残基; 4）测量野生型和 使用GST-融合蛋白体外突变蛋白，并评估 这些之间的关系和亲和力在酵母中证明， 5）构建HSV-1和杆状病毒重组体， 分析它们的依赖ori的复制，并作为筛选 干扰突变; 6）确定蛋白质的层次：蛋白质 在没有DNA的情况下，蛋白质组装过程中的相互作用 使用免疫荧光和共聚焦显微镜进行合成。

Herpes simplex virus (HSV) provides a tractable genetic model system for eukaryotic DNA replication since it encodes at least 7 proteins which are involved directly in the synthesis of viral DNA, all of which are essential for virus replication. Two of these proteins are responsible for elongation of the DNA chain--the DNA polymerase (pol) catalytic core and its accessory protein UL42. These proteins form a stable complex in vitro and together perform processive DNA synthesis. The long term goal of the project is to understand the role of UL42 in DNA replication and how it interacts with pol for processive DNA replication. It is hypothesized that interference with the ability of UL42 to complex with pol will block viral DNA synthesis. Thus, identification of the domains of UL42 and pol which are critical for their interaction provides a target for the development of novel anti-viral compounds. Due to the structural conservation of pol accessory proteins among highly divergent species, it is possible that defining the structure of this complex will lead to an even broader approach for the rational design of compounds capable of inhibiting DNA synthesis in neoplastic cells. The specific aims of the project are 1) to determine the effect of UL42 on the mechanism of DNA polymerization using transient kinetic analysis; 2) to use the yeast two- hybrid system to identify the subdomains of UL42 and pol capable of physical interaction in vivo; 3) to select for non-interacting pol and UL42 mutants and extragenic suppressors in the yeast two-hybrid system following random mutagenesis using PCR or oligo-directed mutagenesis to alter charged residues; 4) to measure the affinities of wild-type and mutant proteins in vitro using GST-fusion proteins and to evaluate the relationship between these and the affinities demonstrated in the yeast two-hybrid system; 5) to construct HSV-1 and baculovirus recombinants and to analyze them for ori-dependent replication and as a means to screen for interference mutations; 6) to determine the hierarchy of protein:protein interactions during the assembly of proteins in the absence of DNA synthesis using immunofluorescence and confocal microscopy.