PROTEINS INVOLVED IN REPLICATION OF HSV DNA

单纯疱疹病毒1型和2型（HSV-1和HSV-2）感染 这是美国的一个主要健康问题。 这项建议旨在 为了纯化和表征存在于HSV感染细胞中的蛋白质， 参与病毒DNA的复制和表达。 我们将 继续我们对主要HSV DNA结合（ICP 8）的研究。 亲和常数 结合的化学计量将获得的ICP 8的相互作用 单链和双链核酸。 我们还将确定 这种蛋白质在核酸之间的交换率。 的各种 ICP 8的功能结构域将通过特异性修饰来定位 通过对ICP 8特性的研究， 在不同位置含有温度敏感损伤的分子。 ICP 8的存在对同源（HSV）和异源（HSV）的影响 （哺乳动物、酵母和原核）DNA聚合酶。 一 在HSV感染的细胞中鉴定出的DNA引发酶活性将 被隔离和特征化。 将使用纯化的引发酶活性 试图重建HSV复制装置 Z-DNA结合 将鉴定HSV系统中的蛋白质和Z-DNA后续序列， 与世隔绝 将检测Z-DNA结合蛋白的结合特异性。 使用克隆的HSV限制性内切酶片段。 Z-DNA序列 将使用chloram测试HSV DNA中存在的增强子活性， 酚转乙酰酶（CAT）测定。 使用的方法将包括： 荧光光谱法，滤膜结合，分子克隆，DNA琼脂糖/ 纤维素层析和Z-DNA免疫亲和测定。

Infections with Herpes Simplex Virus types 1 and 2 (HSV-1 and HSV-2) constitute a major health problem in the United States. This proposal aims to purify and characterize proteins present in HSV infected cells which are involved in the replication and expression of the viral DNA. We will continue our work on the major HSV DNA binding (ICP8). Affinity constants and stoichiometries of binding will be obtained for the interaction of ICP8 with single and double-stranded nucleic acids. We will also determine the rate of exchange of this protein between nucleic acids. The various functional domains of ICP8 will be mapped by means of specific modification of individual amino acid residues and by studies on the properties of ICP8 molecules containing temperature sensitive lesions in various locations. The effect of the presence of ICP8 on homologous (HSV) and heterologous (mammalian, yeast and procaryotic) DNA poloymerases will be determined. A DNA primase activity which has been identified in HSV-infected cells will be isolated and characterized. The purified primase activity will be used in an attempt to reconstruct the HSV replication apparatus. Z-DNA binding proteins and Z-DNA suequences in the HSV system will be identified and isolated. The Z-DNA binding proteins will be tested for binding specifi- city using cloned HSV restriction enzyme fragments. Z-DNA sequences present in HSV DNA will be tested for enhancer activity using a chloram- phenicol transacetylase (CAT) assay. Methodology used will include: fluorescence spectroscopy, filter binding, molecular cloning, DNA agarose/ cellulose chromatography and Z-DNA immunoaffinity assays.