ELUCIDATION OF THE BIOLOGICAL ROLE OF Z-DNA IN E.COLI

旨在阐明Z-DNA的生物学作用的实验 有两种形式。可以形成Z-DNA的自然序列已经被 鉴定和纯化了与Z-更好结合的蛋白质 在体外，DNA比B-DNA更稳定。然而，这些序列还没有被 这些蛋白质在体内形成Z-DNA，也没有显示出 体内结合Z-DNA。我们最近从大肠杆菌中提纯了三种蛋白质 在体外与Z-DNA特异性结合，制备了单抗 抗它们的抗体，并克隆它们的基因。对于第一次 可利用的时间试剂使分子分析成为可能 编码Z-DNA结合蛋白的基因和广泛的生化 对这些蛋白质的研究。这些研究的目标是 通过测定Z-DNA在大肠杆菌中的生物学作用 这些Z-DNA结合蛋白的功能。我们将按顺序排列 并在大肠杆菌图谱上定位它们，并分析 蛋白质在二维速度上的迁移。比较一下这个 有可用的数据库的信息将揭示这些数据 蛋白质或基因已经被收集。使用等位基因替换 并构建了过度表达或产生这些反义mRNA的结构 我们将产生零突变或条件性致死的基因 过度表达这些基因的菌株；或产生水平降低的菌株 这些蛋白质中。我们将检查突变体中是否存在缺陷 在DNA水平上执行的过程：复制、重组、 修复、超级缠绕和转录。如果我们获得有条件的 我们将克隆其他基因中的突变，这些基因可以抑制 原始突变表型。使用高纯度的蛋白质从 过量表达这些基因的菌株我们将进行广泛的 研究这些蛋白质与Z-DNA的相互作用。我们会 精确定量这些蛋白质对Z-DNA的特异性。 化学修饰实验将揭示细节，在基地 交互的配对级别。我们将确定活体内的 免疫共沉淀法测定这些蛋白质的结合部位 细胞萃取物中的连接蛋白-DNA复合体及其分析 通过克隆和测序使DNA沉淀。纯化的蛋白质将 同时检测NTPase、核酸酶、拓扑异构酶和 重组活动。由于对Z-Z的了解如此之少 DNA在细胞中起作用，我们相信大肠杆菌，这是服从于 基因分析是最简单和最具特点的 实验室生物体，是开始研究的理想系统 Z-DNA的功能。这些研究将产生深远的影响 暗示，因为它们将为Z-的功能提供线索 在除大肠杆菌以外的生物中的DNA。

Experiments-aimed at elucidating the biological role of Z-DNA have taken two forms. Natural sequences which can form Z-DNA have been identified and proteins have been purified which bind better to Z- DNA than B-DNA in vitro. However, the sequences have not been shown to form Z-DNA in vivo, nor have the proteins been shown to bind Z-DNA in vivo. We recently purified three proteins from coli which bind specifically to Z-DNA in vitro, prepared monoclonal antibodies against them, and cloned their genes. For the first time reagents are available that make possible molecular analyses of ,genes encoding Z-DNA binding proteins and extensive biochemical studies of these proteins. The goal of these studies is to elucidate the biological role of Z-DNA in E. coli by determining the function of these Z-DNA binding proteins. We will sequence their genes and locate them on the E. coli map, and analyze the migration of the proteins on 2-D PACE. Comparison of this information with available data banks will reveal if data on these proteins or genes has been collected. Using allelic replacement and constructs overexpressing or producing antisense mRNA for these genes we will generate null mutants or conditional lethals and strains which overexpress these genes ;or produce reduced levels of these proteins. We will examine the mutants for defects in processes executed at the DNA level: replication, recombination, repair, supercoiling, and transcription. If we obtain conditional lethals we will clone mutants in other genes which suppress the original mutant phenotype. Using highly purified protein from strains overexpressing these genes we will carry out an extensive study of the interaction of these proteins with Z-DNA. We will precisely quantitate the specificity of these proteins for Z-DNA. Chemical modification experiments will reveal details, at the base pair level, of the interaction. We will identify the in vivo binding sites of these proteins by immunoprecipitating UV cross- linked protein-DNA complexes from cell extracts and analyzing the precipitated DNA by cloning and sequencing. Purified proteins will also be assayed for NTPase, nuclease, topoisomerase, and recombination activities. Since so little is known about what Z- DNA does in a cell, we believe that E. coli, which is amenable to genetic analysis and is one of the simplest and best characterized laboratory organisms, is an ideal system in which to begin studying the function of Z-DNA. These studies will have far reaching implications since they will provide clues to the function of Z- DNA in organisms other than E. coli.