STRUCTURE AND FUNCTION OF UVRABC EXCISION NUCLEASE

核苷酸切除修复是一种DNA修复机制，涉及 双链DNA中受损核苷酸的去除及闭合 由此产生的差距。在大肠杆菌中，由uvrA编码的UVRABC核酸酶， UvrB和uvrC基因，催化紫外线诱导的嘧啶二聚体的去除 (可能还有其他致癌物质诱导的碱基加合物，它们会扭曲DNA 双螺旋)通过水解第8个磷酸二酯键5‘和第4个或 5‘磷酸二酯键3’到修饰的核苷酸上，并去除 得到12-13个核苷酸长的单链DNA片段。这个项目 将进一步表征UVRABC核酸酶和核苷酸切除 在大肠杆菌中的修复机制。 用沉淀法测定UVRABC核酸酶的亚基组成 甘油梯度和凝胶过滤层析。温度 UvrA、uvrB和uvrC蛋白的敏感突变和缺失突变将是 分离的；纯化的突变蛋白将用于定义功能 亚基的结构域及其在两个切口处的作用 反应。UVR蛋白的序列将通过DNA获得 序列分析和预测的二级结构。 将制备包含各种碱基加合物的DNA片段。这个 与这些加合物相关的切口点将通过孵化来确定 UVRABC核酸酶末端标记片段分析 DNA测序凝胶上的反应产物。测定结合的化验， 将制定切开和切除反应的步骤以确定 控制分子间相互作用的热力学和动力学参数 底物中含有该酶及其亚基。分子中的化学基团 与酶接触的底物将通过烷基化来鉴定。 使用定义顺序的衬底的保护和干扰方法 携带单一的嘧啶二聚体。 核苷酸切除修复将在体外使用UVRABC重建 核酸酶、DNA聚合酶I和大肠杆菌DNA连接酶。这个系统将是 用来调查短补丁和长补丁的来源 体内观察及recA蛋白参与长片修复的情况。

Nucleotide excision repair is a DNA repair mechanism that involves the removal of damaged nucleotides from double-stranded DNA followed by closure of the resulting gap. In E. coli, UVRABC nuclease, encoded by the uvrA, uvrB and uvrC genes, catalyzes the removal of UV-induced pyrimidine dimers (and presumably other carcinogen-induced base adducts which distort the DNA double helix) by hydrolyzing the 8th phosphodiester bond 5' and the 4th or 5th phosphodiester bond 3' to the modified nucleotides and removing the resulting 12-13 nucleotide long single-stranded DNA fragment. This project will further characterize UVRABC nuclease and the nucleotide excision repair mechanism in E. coli. Subunit composition of UVRABC nuclease will be determined by sedimentation in glycerol gradients and by gel filtration chromatography. Temperature sensitive and deletion mutants of the uvrA, uvrB and uvrC proteins will be isolated; purified mutant proteins will be used to define the functional domains of the subunits and the role of each in the two incision reactions. The sequences of the uvr proteins will be obtained by DNA sequence analysis and the secondary structures predicted. DNA fragments containing various base adducts will be prepared. The incision sites relative to these adducts will be determined by incubating terminally labeled fragments with UVRABC nuclease and analyzing the reaction products on DNA sequencing gels. Assays measuring the binding, incision, and excision steps of the reaction will be developed to determine the thermodynamic and kinetic parameters governing the interaction of the substrate with the enzyme and its subunits. The chemical groups of the substrate in contact with the enzyme will be identified by alkylation protection and interference methods using a substrate of defined sequence carrying a single pyrimidine dimer. Nucleotide excision repair will be reconstituted in vitro using UVRABC nuclease, DNA polymerase I and E. coli DNA ligase. This system will be used to investigate the origin of the short and long repair patches observed in vivo and the involvement of recA protein in long patch repair.