Structural Biology of Translesion DNA Synthesis

DNA聚合酶催化细胞DNA的复制，在DNA修复中起着不可或缺的作用。 跨损伤合成，即跨受损DNA核苷酸的DNA合成，是 突变和癌症的发展。已经在人类身上发现了十多种能够合成DNA的蛋白质。这些DNA聚合酶在保真度和执行跨损伤合成的能力上有所不同。这个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，这些聚合酶准备进行跨损伤合成。这些结构将允许检验一般假设，即DNA聚合酶活性部位的相互作用调节跨损伤合成的错误编码和/或效率。具体目标涉及以下方面的结构和动力学机制的研究：(A)高保真DNA聚合酶(A、B和X家族)容易出错的跨病变合成；(B)融合受阻的DNA合成；以及(C)专业跨病变合成的准确和容易出错的合成 聚合酶(Y家族)。X射线结晶学将被用来捕捉在这一过程的各个步骤中与氧化损伤的DNA络合的DNA聚合酶。结合稳态动力学分析和定点突变，这些结构应该揭示低(跨损伤)和高保真DNA聚合酶之间的结构和功能差异。高保真聚合酶的结构研究将为dCTP或dATP相对于8-oxo-dG的优先掺入提供洞察，建立一个结构 A规则的基础，并解释了epsilon-DC和其他外环DNA加合物的错误编码潜力。碱性位点和胸腺嘧啶二醇阻止DNA聚合酶进展的机制也将被研究。对两种不同的Y家族(跨损伤)DNA聚合酶，小鼠polKappa和人polETA的结构分析，将提供对它们底物特异性的深入了解。这个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突变的研究(项目1)、热力学参数的确定(项目3)和含有位点特异性的DNA的核磁共振研究 放置的损伤(项目4)。总体而言，该项目提供了与 突变与人类癌症的结构生物学。

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.