Mechanisms of Damaged DNA Replication in Eukaryotes

项目摘要/摘要 突变在许多疾病的病因学中起着基本的作用，包括癌症、糖尿病、 癫痫、精神分裂症和其他疾病。这些突变中的许多都是跨病变合成的结果， 细胞在DNA复制过程中用来绕过DNA损伤的一条途径。在跨损伤中 合成，高保真的经典DNA聚合酶(即，参与正常DNA的聚合酶 复制)遇到DNA损伤和停滞。低保真的非经典聚合酶有 被招募到停滞不前的复制叉子上，在那里它们复制受损的DNA。 每一种非经典聚合酶都专门结合几种不同类型的核苷酸。 DNA损伤的可能性。当使用适当的非经典聚合酶时，损伤旁路不是 致突变性。如果使用了不适当的方法，它就会发生突变。建议的总体目标是 研究是为了了解影响经病变检查的准确性和效率的因素。 综合。我们特别感兴趣的是研究非经典聚合酶如何 在跨损伤合成过程中进行选择和调节。人们对非古典音乐知之甚少 聚合酶选择和调节，因为这些聚合酶在动态的， 难以研究的多蛋白质复合体(旁路复合体)。我们正在使用一种 生化、生物物理和结构方法的创新和新颖组合 将使我们能够克服这些挑战。在目标1中，我们将研究旁路的体系结构 采用单分子全内反射荧光(TIRF)实验。在……里面 目的2，我们将研究稳态和预稳状态下的旁路复合波的调节 状态动力学研究。在目标3中，我们将使用X射线检查旁路复合体的结构 结晶学、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.