Structural Biology of Translesion DNA Synthesis

DNA聚合酶催化细胞DNA的复制，并在DNA修复中发挥不可或缺的作用。 转座合成，即跨受损DNA核苷酸的DNA合成，是DNA合成的关键步骤。 突变和癌症的发展。在人类中已经鉴定出十多种能够合成DNA的蛋白质。这些DNA聚合酶在它们的保真度和进行跨损伤合成的能力方面不同。该项目的目标是解决来自四个不同家族的DNA聚合酶的三维结构，以进行跨损伤合成。这些结构将允许检查的一般假设，即在DNA聚合酶活性位点的相互作用调节的错误编码和/或效率的translesion合成。具体目标包括研究（a）高保真DNA聚合酶（A、B和X家族）的易错跨损伤合成（B）的结构和动力学机制;（b）损伤阻断的DNA合成;（c）特异跨损伤合成的准确和易错合成 聚合酶（Y家族）。X射线晶体学将用于捕获在该过程的各个步骤中与氧化损伤的DNA复合的DNA聚合酶。再加上稳态动力学分析和定点突变，这些结构应该揭示低（translesion）和高保真DNA聚合酶之间的结构和功能差异。使用高保真聚合酶的结构研究将提供对dCTP或dATP相对于8-氧代-dG的优先掺入的洞察，建立结构性的聚合酶。 “A规则”的基础，并解释epsilon-dC和其他环外DNA加合物的错误编码潜力。脱碱基位点和胸腺嘧啶乙二醇阻断DNA聚合酶进展的机制也将被研究。两种不同的Y-家族（translesion）DNA聚合酶，小鼠pol Kappa和人类pol eta的结构分析，将提供洞察其底物特异性。该项目将在原子水平上分析跨损伤合成的机制，并将补充错误编码和诱变的研究（项目1），热力学参数的测定（项目3）和含有位点特异性的DNA的NMR研究。 放置病变（项目4）。总的来说，该项目提供了有关的关键知识， 诱变和人类癌症结构生物学。

DNA polymerases catalyze the replication of cellular DNA and play an integral role in DNA repair. Translesion synthesis, i.e. DNA synthesis across damaged DNA nucleotides, is a critical step in mutagenesis and the development of cancer. More than ten proteins capable of DNA synthesis have been identified in humans. These DNA polymerases differ in their fidelity and ability to perform translesion synthesis. The goal of this project is to solve the three dimensional structure of DNA polymerases from four different families poised to perform translesion synthesis. These structures will allow examination of the general hypothesis that interactions in the DNA polymerase active site modulate the miscoding and/or efficiency of translesion synthesis. The specific aims involve studies of structural and kinetic mechanisms of (a) error-prone translesion synthesis by high fidelity DNA polymerases ( Families A, B and X) (b) Iesion-blocked DNA synthesis and (c) accurate and error-prone synthesis by specialized translesion synthesis polymerases (Family Y). X-ray crystallography will be used to capture DNA polymerases complexed with oxidatively damaged DNA during various steps in this process. Coupled with steady-state kinetic analysis and site-directed mutagenesis, these structures should reveal structural and functional differences between Iow-(translesion) and high-fidelity DNA polymerases. Structural studies with high-fidelity polymerases will provide insight into the preferential incorporation of dCTP or dATP opposite 8-oxo-dG, establish a structural basis for the "A-rule", and explain the miscoding potential of epsilon-dC and other exocyclic DNA adducts. The mechanism by which abasic sites and thymine glycol block the progression of DNA polymerases will also be examined. Structural analysis of two different Y-family (translesion) DNA polymerases, mouse pol Kappa and human pol eta, will provide insight into their substrate specificity. This project will analyze the mechanism of translesion synthesis at the atomic level and will complement studies of miscoding and mutagenesis (Project 1), the determination of thermodynamic parameters (Project 3) and NMR studies on DNA containing site-specifically placed lesions (Project 4). Overall, this project provides critical knowledge relevant to the structural biology of mutagenesis and human cancer.