STRUCTURE AND FUNCTION OF UVRABC EXCISION NUCLEASE

核苷酸切除修复是一种DNA修复机制， 从双链DNA中去除受损的核苷酸，然后闭合 由此产生的差距。 在大肠大肠杆菌，UVRABC核酸酶，由uvrA编码， uvrB和uvrC基因，催化UV诱导的嘧啶二聚体的去除 (and可能是其他致癌物诱导的碱基加合物， 双螺旋）通过水解第8个磷酸二酯键5'和第4个或 将第5个磷酸二酯键3'连接到修饰的核苷酸上，并去除修饰的核苷酸。 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 E.杆菌 UVRABC核酸酶的亚基组成将通过沉降法测定 在甘油梯度中和通过凝胶过滤色谱法。 温度 uvrA、uvrB和uvrC蛋白的敏感突变体和缺失突变体将被 分离的;纯化的突变蛋白将用于定义功能性的 亚基的结构域以及每个亚基在两个切口中的作用 反应. 紫外线蛋白的序列将通过DNA测序获得。 序列分析和二级结构预测。 将制备含有各种碱基加合物的DNA片段。 的 与这些加合物相关的切口部位将通过孵育 用UVRABC核酸酶末端标记的片段，并分析 DNA测序凝胶上的反应产物。 测定结合的测定法， 切口和切除步骤的反应将制定，以确定 热力学和动力学参数控制的相互作用， 底物与酶及其亚基。 的化学基团， 与酶接触的底物将通过烷基化鉴定 使用确定序列的底物的保护和干扰方法 带有一个嘧啶二聚体。 核苷酸切除修复将使用UVRABC在体外重建 核酸酶、DNA聚合酶I和E. coli DNA连接酶。 该系统将 用于研究短和长修复斑块的起源 在体内观察到recA蛋白参与长斑修复。

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.