Coordination of DNA replication, repair, and translesion DNA synthesis

DNA 复制、修复和跨损伤 DNA 合成的协调

基本信息

批准号：
8630539
负责人：
MARK D. SUTTON
金额：
$ 34.79万
依托单位：
STATE UNIVERSITY OF NEW YORK AT BUFFALO
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-05-01 至 2017-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8630539
关键词：
Address Aging Antibiotic Resistance Antibiotics Archaea Area Back Bacteria Bacterial DNA Biochemical Biochemical Genetics Biological Assay Cell physiology Cells Collaborations Complex DNA DNA Damage DNA Polymerase III DNA Polymerase beta DNA Repair DNA biosynthesis DNA-Directed DNA Polymerase Decision Making E coli replicase Escherichia coli Eukaryota Exposure to Failure Genetic Genomic Instability Genomics Goals Grant Human Immune response In Vitro Individual Knowledge Malignant Neoplasms Measures Modeling Molecular Molecular Models Molecular Sieve Chromatography Mutagenesis Mutation Organism Pathway interactions Play Polymerase Primer Extension Process Proteins Pseudomonas aeruginosa Reactive Oxygen Species Regulation Research Roentgen Rays Role Slide Testing Therapeutic Vertebral column Virulence Work base cell growth cystic fibrosis airway defined contribution human disease insight light scattering mange molecular modeling novel pathogen pathogenic bacteria public health relevance repaired research study single molecule tool

项目摘要

DESCRIPTION (provided by applicant): Failure to efficiently coordinate DNA replication with other cellular processes results in mutations and genome instability, contributing to numerous human disease states, including cancers. Mutations in human pathogens, particularly those caused by reactive oxygen species (ROS) generated by the host immune response, or exposure to antibiotics, promote their adaptation to the host (i.e., pathoadaptation), exacerbating treatment. The long-term goal of our research is to develop an integrated mechanistic understanding of how organisms coordinate the actions of their DNA replication machinery with those of other cellular factors that act in DNA repair and damage tolerance. Work in our lab over the last 10 years supported by this grant has had a major impact on our understanding of mechanisms coordinating the actions of the E. coli replicase with those of translesion DNA synthesis DNA polymerases (TLS Pols). Our findings successfully challenged the well- established tool belt model. We have also shown that errors catalyzed by Pseudomonas aeruginosa DNA polymerase IV (Pol IV) contribute to mutations that likely promote persistence of this pathogen in cystic fibrosis airways. A molecular understanding of the mechanisms that contribute to mutations is crucial to our understanding of the basis for genome instability, human disease, and pathoadaptation, as well as efforts to develop novel therapies. The proposed research addresses unanswered questions regarding mechanisms that organisms use to manage the actions of their diverse Pols. We will focus our efforts in two critical yet understudied areas. During the prior period of support, we discovered that specific E. coli beta-clamp-DNA interactions are required for DNA damage-induced mutagenesis, suggesting they impart a hierarchical order to Pol switches that may be exploited to control mutation rate. In Aim 1, we will determine the contributions of the different beta-clamp-DNA interactions to replication fidelity and TLS using a combination of genetic, biochemical, biophysical, and single molecule approaches. In Aim 2, we will use small angle X-ray scattering (SAXS), size exclusion chromatography-multi angle light scattering (SEC-MALS), molecular modeling, and biochemical approaches to structurally define complexes consisting of the 5 different E. coli Pols, clamp, and DNA. Using insights gained from these efforts, together with genetic, biochemical, biophysical, and single molecule approaches, we will define the mechanisms by which E. coli Pols switch. We will also determine whether an ability to impede Pol III processivity is shared by other proteins that switch with Pol III. Results from these experiments will provide unprecedented insight into the molecular mechanisms underlying coordinate regulation of DNA replication, DNA repair, and TLS. Furthermore, we anticipate that our results will identify critical steps in these evolutionarily conserved processes that can be targeted to control proficiency and fidelity of replication for therapeutic gain.

描述（由申请人提供）：未能有效协调 DNA 复制与其他细胞过程会导致突变和基因组不稳定，从而导致多种人类疾病状态，包括癌症。人类病原体的突变，特别是由宿主免疫反应产生的活性氧（ROS）或接触抗生素引起的突变，会促进它们对宿主的适应（即病理适应），从而加剧治疗。我们研究的长期目标是对生物体如何协调其 DNA 复制机制与其他参与 DNA 修复和损伤耐受的细胞因子的作用建立一个综合的机制理解。过去 10 年，我们实验室在这项资助的支持下所做的工作对我们对大肠杆菌复制酶与跨损伤 DNA 合成 DNA 聚合酶 (TLS Pols) 的作用协调机制的理解产生了重大影响。我们的研究结果成功挑战了完善的工具带模型。我们还表明，铜绿假单胞菌 DNA 聚合酶 IV (Pol IV) 催化的错误会导致突变，从而可能促进这种病原体在囊性纤维化气道中的持续存在。对突变机制的分子理解对于我们理解基因组不稳定性、人类疾病和病理适应的基础以及开发新疗法的努力至关重要。拟议的研究解决了有关生物体用来管理其不同波尔斯行为的机制的尚未解答的问题。我们将把我们的努力集中在两个关键但尚未得到充分研究的领域。在前期支持期间，我们发现特定的 E. DNA 损伤诱导的突变需要大肠杆菌 beta-clamp-DNA 相互作用，这表明它们为 Pol 开关赋予了分层顺序，可用于控制突变率。在目标 1 中，我们将结合遗传、生物化学、生物物理和单分子方法来确定不同的 β-clamp-DNA 相互作用对复制保真度和 TLS 的贡献。在目标 2 中，我们将使用小角 X 射线散射 (SAXS)、尺寸排阻色谱-多角度光散射 (SEC-MALS)、分子建模和生化方法来从结构上定义由 5 种不同的大肠杆菌聚合物组成的复合物、钳子和DNA。利用从这些努力中获得的见解，再加上遗传、生物化学、生物物理和单分子方法，我们将定义大肠杆菌 Pols 转换的机制。我们还将确定与 Pol III 转换的其他蛋白质是否具有阻碍 Pol III 持续进行能力的能力。这些实验的结果将为 DNA 复制、DNA 修复和 TLS 协调调控的分子机制提供前所未有的见解。此外，我们预计我们的结果将确定这些进化保守过程中的关键步骤，这些步骤可以有针对性地控制复制的熟练程度和保真度以获得治疗效果。