VIRAL AND PLASMID DNA, PROTEIN INTERACTIONS

这项研究旨在将真核病毒牛痘用于 几种酶的作用机理和结构的研究 含有DNA的蛋白质。DNA代谢中可能起作用的三种酶 将进行详细研究，所有这些都是针对病毒的。这些措施包括 I型拓扑异构酶(TOPO I)、II型拓扑异构酶(TOPO II)和 一种催化切割和交联链的酶 病毒DNA(缺口连接酶)。这些酶都是DNA结合的 蛋白质，所有这些都存在于纯化的病毒颗粒中。Topo I有 以前被提纯到同质。中的共价中间体 与DNA的反应将被鉴定和分离，并将序列 确定了结合部位的特异性。单抗将会是 这些信息将被用来评估其在 转录和DNA复制。Topo II与划线连接 利用亲和层析和亲和层析将酶纯化到均一 凝胶过滤。纯化的酶的物理性质将是 有文件记录的与DNA共价的中间体将被分离，并 捆绑和切割的特异性将被确定。环境问题 将确定对催化有重要意义的变量，包括在 离子强度、温度和pH；特定离子；以及辅因子。 将获得针对这些酶的单抗，就像在 I型拓扑异构酶，用于研究它们在转录中的作用 和DNA复制。将绘制I型病毒topo基因的图谱，使用 杂交选择基因的体外翻译及两种酶的检测 活性和免疫沉淀。除了这三种酶， 之前对两种病毒DNA结合结构蛋白的研究将 待续。已经获得了针对这两种病毒的抗血清，11K 道尔顿蛋白和24K道尔顿蛋白。11K蛋白将被定位在 痘苗病毒基因组，最终目标是绘制晚期 启动子序列。24K的物理和DNA结合特性 多肽将被测定。与超螺旋DNA的结合 增加超卷将被用来评估 结合到DNA的天然和变性形式。这些实验将 有助于为了解这种病毒的形态发生奠定基础， 其宿主细胞系起源于哺乳动物。

This research is designed to employ the eucaryotic virus vaccinia for the examination of the mechanism of action of several enzymes and structural proteins with DNA. Three enzymes with a putative role in DNA metabolism will be studied in detail, all of them being virus-specific. These include a type I topoisomerase (topo I), a type II topoisomerase (topo II), and an enzyme which catalyzes the nicking and crosslinking of the strands of the viral DNA (nicking-joining enzyme). These enzymes are all DNA binding proteins, and all are present in purified virus particles. Topo I has previously been purified to homogeneity. Covalent intermediates in the reaction with DNA will be identified and isolated and the sequence specificity of the binding site determined. Monoclonal antibodies will be obtained against the enzyme and these will be used to assess its role in transcription and in DNA replication. Topo II and the nicking-joining enzyme will be purified to homogeneity, using affinity chromatography and gel filtration. The physical properties of the purified enzymes will be documented, covalent intermediates with DNA will be isolated, and site specificity of binding and incision will be ascertained. The environmental variables significant for catalysis will be determined, including optima in ionic strength, temperature, and pH; specific ions; and cofactors. Monoclonal antibodies will be obtained against these enzymes and, as with the type I topoisomerase, used to investigate their roles in transcription and DNA replication. The viral type I topo gene will be mapped, using in vitro translation of hybrid-selected mRNA and detection by both enzyme activity and immunoprecipitation. In addition to these three enzymes, previous investigations of two viral DNA-binding structural proteins will be continued. Antisera have been obtained against both of these, an 11K dalton protein and a 24K dalton protein. The 11K protein will be mapped in the vaccinia genome, with the eventual objective of mapping the late promoter sequences. The physical and DNA-binding properties of the 24K polypeptide will be determined. The binding to superhelical DNA of increasing supercoiling will be used to assess the relative strength of binding to the native and denatured forms of DNA. These experiments will help lay a foundation for understanding the morphogenesis of this virus, the host cell lines of which are of mammalian origins.