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中枢事件中的作用 病毒（HSV-1）复制。 初步实验， 这强烈表明病毒拓扑异构酶活性与 65,000道尔顿的延迟早期基因产物。 该提案基于 发展分析系统，使其能够测量 HSV-1拓扑异构酶活性在同源背景下的选择性 宿主酶 此外，充分表征的单特异性抗体 针对p65的基因将用于绘制基因图谱， 编码p65并确定转录方向。 更精确 转录本的映射将被执行，目的是识别 5'和3'末端。 最后，蛋白质编码以及向上和向下， 下游区域将通过DNA测序进行分析。 该项目的下一步是利用基因中的信息 水平来严格检验基因产物 编码p65的是拓扑异构酶。 这一目标将通过 在基因中构建突变并评估所得基因 使用对p65特异性的拓扑异构酶测定的产物 相关酶 最后，将大力致力于 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提高我们对 拓扑异构酶的作用，在必要的遗传过程中，伴随 HSV-1的复制。 拓扑异构酶切割并共价结合病毒 因此，对这些独特的DNA结合蛋白的研究是重要的 因为抗病毒化学疗法的新途径将被打开。

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.