Mechanisms of Damaged DNA Replication in Eukaryotes

真核生物中受损 DNA 复制的机制

基本信息

批准号：
10004053
负责人：
M. TODD WASHINGTON
金额：
$ 33.29万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-07-01 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10004053
关键词：
Address Adopted Affect Architecture Binding Binding Sites Biochemical Biophysics Bypass Catalytic Domain Cell Death Cells Complex DNA DNA Damage DNA Polymerase I DNA Replication Damage DNA biosynthesis DNA replication fork DNA-Directed DNA Polymerase Diabetes Mellitus Disease Enzymes Epilepsy Etiology Eukaryota Fluorescence Fluorescence Resonance Energy Transfer Genome Stability Genomic Instability Goals Individual Kinetics Lead Lesion Malignant Neoplasms Measurement Molecular Conformation Multiprotein Complexes Mutation Nucleotides Pathway interactions Play Polymerase Process Proteins Regulation Research Resolution Roentgen Rays Role Schizophrenia Structure Testing Ubiquitin Work Wrist X-Ray Crystallography base disease-causing mutation experimental study flexibility innovation interest mutant novel novel strategies recruit simulation single molecule tool

项目摘要

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.

项目摘要/摘要突变在许多疾病的病因中起着基本作用，包括癌症，糖尿病，癫痫，精神分裂症等。这些突变中的许多突变是由跨性别合成引起的该细胞在DNA复制过程中绕过DNA损伤的途径。在Translesion中合成，高保真经典的DNA聚合酶（即，与正常DNA有关复制）遇到DNA损伤和失速。低保真非经典聚合酶是被招募到停滞的复制叉，在那里对受损的DNA进行复制。每个非古典聚合酶均专门掺入几种类型的核苷酸 DNA损伤。当使用适当的非古典聚合酶时，损坏旁路不是诱变。当使用不正确的时，它是诱变的。拟议的总体目标研究是了解影响跨性别的准确性和效率的因素合成。我们特别有兴趣研究非古典聚合酶在跨性别合成过程中选择并调节。关于非古典的知之甚少聚合酶选择和调节，因为这些聚合酶在动态中起作用，难以研究的多蛋白质复合物（旁路复合物）。我们正在使用生化，生物物理和结构方法的创新和新颖的组合将使我们能够克服这些挑战。在AIM 1中，我们将检查旁路的架构使用单分子总内反射荧光（TIRF）实验的复合物。在 AIM 2，我们将使用稳态和稳态检查旁路复合物的调节州动力学研究。在AIM 3中，我们将使用X射线检查旁路复合物的结构晶体学，小角度X射线散射（SAXS）和布朗动力学（BD）模拟。