Mechanisms of Damaged DNA Replication in Eukaryotes

项目总结/摘要 突变在许多疾病的病因学中起着重要作用，包括癌症，糖尿病， 癫痫精神分裂症等等这些突变中的许多是跨损伤合成的结果， 这是细胞在DNA复制过程中用来绕过DNA损伤的一种途径。经病变 合成，高保真经典DNA聚合酶（即，与正常DNA有关的基因 复制）遇到DNA损伤和停滞。低保真非经典聚合酶是 被招募到停滞的复制叉上，在那里它们进行受损DNA的复制。 每一种非经典的聚合酶都是专门用于整合与几种类型相反的核苷酸的 DNA损伤。当使用适当的非经典聚合酶时， 致突变的当使用不当时，它是致突变的。拟议的总体目标 研究的目的是了解影响经皮穿刺准确性和效率的因素， 合成.我们特别感兴趣的是研究非经典聚合酶是如何 在跨损伤合成期间被选择和调节。对非经典的知之甚少 聚合酶的选择和调节，因为这些聚合酶在动态， 多蛋白复合物（旁路复合物），这是很难研究。我们正在使用 生物化学、生物物理和结构方法的创新和新颖组合， 将帮助我们克服这些挑战。在目标1中，我们将研究旁路的架构 复合物使用单分子全内反射荧光（TIRF）实验。在 目的2，我们将研究使用稳态和预稳态的旁路复合物的调节 状态动力学研究。在目标3中，我们将使用X射线检查旁路复合物的结构 晶体学、小角X射线散射（SAXS）和布朗动力学（BD）模拟。

Project Summary/Abstract Mutations play a fundamental role in the etiology of many diseases including cancers, diabetes, epilepsy, schizophrenia, and others. Many of these mutations result from translesion synthesis, a pathway the cell uses to bypass DNA damage during DNA replication. In translesion synthesis, the high-fidelity classical DNA polymerase (i.e., the one involved in normal DNA replication) encounters DNA damage and stalls. Low-fidelity non-classical polymerases are recruited to the stalled replication fork where they carry out replication of the damaged DNA. Each non-classical polymerase is specialized for incorporating nucleotides opposite a few types of DNA damage. When the proper non-classical polymerase is used, damage bypass is not mutagenic. When an improper one is used, it is mutagenic. The overall goal of the proposed research is to understand the factors that affect the accuracy and efficiency of translesion synthesis. We are particularly interested in examining how non-classical polymerases are selected and regulated during translesion synthesis. Little is known about non-classical polymerase selection and regulation, because these polymerases function within dynamic, multi-protein complexes (bypass complexes) that are difficult to study. We are using an innovative and novel combination of biochemical, biophysical, and structural approaches that will allow us to overcome these challenges. In Aim 1, we will examine the architecture of bypass complexes using single-molecule total internal reflection fluorescence (TIRF) experiments. In Aim 2, we will examine the regulation of bypass complexes using steady state and pre-steady state kinetics studies. In Aim 3, we will examine the structure of bypass complexes using X-ray crystallography, small-angle X-ray scattering (SAXS) and Brownian dynamics (BD) simulations.