Nucleotide and Base Excision Repair

该项目(核苷酸切除修复和碱基切除修复)的重点是 了解关键蛋白质与DNA底物和对功能至关重要的伙伴蛋白质的相互作用 DNA切除修复复合体。我们的目标是利用修复的结构和生化研究 用ERCC1-XPF内切酶和DNA连接酶解释多步反应的配位性 核苷酸切除修复(NER)与B_ase切除修复的完成 (NER)。DNA切除修复蛋白和途径的缺陷会导致突变率增加， 染色体断裂，癌症发病率增加。DNA螺旋结构的扭曲 是由修复酶特定识别的，并且可以不依赖于 损害的化学特征。这一普遍的策略使一种酶能够启动一种 DNA中的各种损伤。此外，与其他DNA结合和修复蛋白的相互作用提供 额外的生物特异性和有助于修复的效率。尽管酶的活性 基本的NER和BER通路的构成是已知的，但这些活动是如何进行的还有待确定 通过酶-DNA内部的物理相互作用协调成多步反应途径 催化DNA损伤切除修复的复合体。相关酶-DNA的结构分析 复合体将揭示酶及其相应的DNA底物的不同构象状态 修复反应的不同步骤。来自X射线的低分辨率结构和构象 在溶液中的散射将补充修复复合体的高分辨率图像， 结晶了。通过这种方式，催化DNA的多蛋白质复合体的动态组装和拆解 维修将以此为特征。我们建议通过研究特定的案例来检验和发展这些假设。 切除修复成分如下：1)XPF-ERCC1及其相关DNA的催化底物专一性 结构特异性核酸酶；2)XPA将XPFERCC1招募到NER复合体的损伤感觉；3)DNA连接酶的催化选择性；缺口传感和DNA修复；4)DNA连接酶I与DNA滑动夹的相互作用；以及5)连接酶I与夹带加载器的相互作用。

The Project (Nucleotide Excision Repair and Base Excision Repair) focuses on understanding the interactions of key proteins with DNA substrates and partner proteins critical for functional DNA excision repair complexes. Our goal is to leverage structural and biochemical studies of the repair endonuclease ERCC1-XPF and of DNA ligase to illuminate the coordination of the multi-step reaction pathways that comprise Nucleotide Excision Repair (NER) and the completion of B_ase Excision Repair (NER). Defects in DNA excision repair proteins and pathways result in increased rates of mutation, chromosomal breakage, and an increased incidence of cancers. Distortions of the helical structure of DNA are specifically recognized by repair enzymes and can be read out in a way that does not depend on the chemical identity of the damage. This generalized strategy enables one enzyme to initiate the repair of a variety of lesions in DNA. Moreover, interactions with other DNA binding and repair proteins provide additional biological specificity and contribute to the efficiency of repair. Although the enzymatic activities constituting the basic NER and BER pathways are known, it remains to be determined how these activities are coordinated into a multi-step reaction pathway by the physical interactions within enzyme-DNA complexes catalyzing the excision repair of DNA damage. Structural analyses of the relevant enzyme-DNA complexes will reveal distinct conformational states of the enzymes and their DNA substrates corresponding to different steps of the repair reaction. Low-resolution structures and conformations derived from x-ray scattering in solution will complement high-resolution images of the repair complexes that can be crystallized. In this way, the dynamic assembly and disassembly of multi-protein complexes catalyzing DNA repair will be characterized. We propose to test and develop these hypotheses by investigating specific excision repair components as follows: 1) Catalytic Substrate specificity of XPF-ERCC1, and related DNA structure-specific nucleases; 2) Damage senses by XPA that recruits XPFERCC1 to NER complexes; 3) Catalytic selectivity of DNA ligases; Nick-sensing and DNA repair; 4) Interactions of DNA Ligase I with DNA sliding clamps; and 5) Interactions of ligase I with the clamp loaders.