VIRAL AND PLASMID DNA, PROTEIN INTERACTIONS

本研究旨在利用真核病毒牛痘， 检查几种酶的作用机制和结构 蛋白质与DNA 在DNA代谢中具有假定作用的三种酶 将被详细研究，所有这些都是病毒特异性的。 这些包括 I型拓扑异构酶（topo I）、II型拓扑异构酶（topo II）和 酶，催化切割和交联的链， 病毒DNA（切口连接酶）。 这些酶都与DNA结合 蛋白质，并且全部存在于纯化的病毒颗粒中。 Topo I有 之前被纯化至均匀。 中间体中的共价键 与DNA的反应将被识别和分离， 确定结合位点的特异性。 单克隆抗体将是 获得的酶，这些将用于评估其作用， 转录和DNA复制。 Topo II和切口连接 使用亲和层析将酶纯化至均一， 凝胶过滤。 纯化的酶的物理性质将是 将分离有记录的与DNA的共价中间体，并研究中心 将确定结合和切割的特异性。 环境 将确定对催化作用有意义的变量，包括最佳的 离子强度、温度和pH;特定离子;和辅因子。 将获得针对这些酶的单克隆抗体， I型拓扑异构酶，用于研究它们在转录中的作用 DNA复制。 I型病毒的拓扑基因将被定位， 杂交选择mRNA的体外翻译和通过两种酶的检测 活性和免疫沉淀。 除了这三种酶， 以前对两种病毒DNA结合结构蛋白的研究将 请继续。 已经获得了针对这两种病毒的抗血清，即11 K 道尔顿蛋白和24 K道尔顿蛋白。 11 K蛋白将在 牛痘基因组，最终目标是绘制晚期 启动子序列。 24 K的物理和DNA结合特性 多肽将被确定。 与超螺旋DNA的结合 增加超螺旋将用于评估 与天然和变性形式的DNA结合。 这些实验将 为了解这种病毒的形态发生奠定了基础， 其宿主细胞系是哺乳动物来源的。

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.