FUNCTIONAL ANALYSIS OF DNA TOPOISOMERASE I IN HSV

这项工作的目的是为了更好地了解 DNA拓扑异构酶在单纯疱疹病毒中枢事件中的作用 病毒(HSV-1)复制。给出了初步的实验结果 有力地表明，病毒的拓扑异构酶活性与 延迟了6.5万道尔顿的早期基因产物。这项提议是基于 化验系统的发展，使其能够测量 HSV-1拓扑异构酶在同源背景下的选择性活性 宿主酶。此外，特性良好的单特异性抗体 针对p65基因将其用于作图，鉴定出该基因的mRNA 编码p65并确定转录方向。更精确 将对成绩单进行测绘，目的是确定 5‘和3’末端。最后，编码蛋白质以及UP和UP 下游区域将通过DNA测序进行分析。 该项目的下一步是利用从基因中获得的信息 水平来严格测试基因产物的假设 编码p65是一种拓扑异构酶。这一目标将通过以下方式实现 构建基因突变并评估产生的基因 使用特异性p65的拓扑异构酶分析的产物 伴生酶。最后，将在以下方面作出重大努力 确定p65是病毒中的必需基因还是非必需基因 体外复制周期。 这项研究的主要意义是增进我们对 拓扑异构酶在伴随而来的基本遗传过程中的作用 单纯疱疹病毒1型的复制。拓扑异构酶裂解和共价结合病毒 因此，对这些独特的DNA结合蛋白的研究是重要的 因为抗病毒化疗的新途径将被开辟。

The objective of this work is to gain a better understanding of the functional role of DNA topoisomerases in central events of herpes simplex virus (HSV-1) replication. Preliminary experiments are presented which strongly indicate that a viral topoiosomerase activity is associated with a delayed early gene product of 65,000 daltons. The proposal is based upon the development of assay systems which make it possible to measure the HSV-1 topoisomerase activity selectively against a background of cognate host enzymes. In addition, well characterized monospecific antibody directed against p65 will be used in mapping the gene, identifying the mRNA encoding p65 and establishing the direction of transcription. More precise mapping of the transcript will be performed with the purpose of identifying 5' and 3' termini. Finally, the protein coding as well as up and downstream regions will be analyzed by DNA sequencing. The next step of the project is to use the information derived at the gene level to rigorously test the hypothesis that the product of the gene encoding p65 is a topoisomerase. This goal will be achieved by constructing mutations in the gene and evaluating the resulting gene product using the topoisomerase assays which are specific the p65 associated enzyme. Finally, a major effort will be devoted toward establishing if p65 is an essential or non-essential gene in the viral replication cycle in vitro. The primary significance of this research is to advance our knowledge of the role of topoisomerases in essential genetic processes that accompany the replication of HSV-1. Topoisomerases cleave and covalently bind viral DNA, therefore studies of these unique DNA binding proteins are important since new avenues for antiviral chemotherapy will be opened.