Coordination of DNA replication, repair, and translesion DNA synthesis

DNA 复制、修复和跨损伤 DNA 合成的协调

• 批准号: 9041875
• 负责人: MARK D. SUTTON
• 金额: $ 0.99万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 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/9041875
• 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; Goals; Grant; Health; 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; biophysical techniques; cell growth; cystic fibrosis airway; defined contribution; genome integrity; human disease; insight; light scattering; mange; molecular modeling; novel; pathogen; pathogenic bacteria; repaired; research study; single molecule; tool

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）：未能有效地协调DNA复制与其他细胞过程导致突变和基因组不稳定性，这导致了包括癌症在内的许多人类疾病状态。人类病原体中的突变，特别是由宿主免疫反应或暴露于抗生素产生的活性氧（ROS）引起的突变，促进了它们对宿主的适应性（即病态加热），加剧治疗。我们研究的长期目标是对生物如何将其DNA复制机制的作用与其他细胞因子的作用进行综合的机械理解，以使其在DNA修复和损伤耐受性中作用。在过去的十年中，这笔赠款支持的工作对我们对机制的理解产生了重大影响，该机制将大肠杆菌复制酶与Translesion DNA合成DNA聚合酶（TLS POLS）的作用相结合。我们的发现成功挑战了良好的工具带模型。我们还表明，铜绿假单胞菌DNA聚合酶IV（POL IV）催化的误差有助于突变，这些突变可能会促进这种病原体在囊性纤维化气道中的持久性。对有助于突变的机制的分子理解对于我们对基因组不稳定性，人类疾病和病原体的基础的理解以及开发新疗法的努力至关重要。拟议的研究涉及有关生物体用来管理其各种pol行动的机制的未解决的问题。我们将把精力集中在两个关键但经过研究的领域。在前期的支持期间，我们发现了特定的E。 DNA损伤诱导的诱变所需的大肠杆菌β-斜线-DNA相互作用是必需的，这表明它们为可以利用的POL开关赋予了分层顺序，这些开关可以利用控制突变速率。在AIM 1中，我们将使用遗传，生化，生物物理和单分子方法的组合来确定不同β-斜线-DNA相互作用对复制保真度和TLS的贡献。在AIM 2中，我们将使用小角度X射线散射（SAX），尺寸排除色谱 - 形式 - 呈现角光散射（SEC-MALS），分子建模和生化方法来定义由5种不同的大肠杆菌Pol，CLAMP和DNA组成的结构定义复合物。利用从这些努力中获得的见解，以及遗传，生化，生物物理和单分子方法，我们将定义大肠杆菌切换的机制。我们还将确定是否能够阻碍使用POL III的其他蛋白质共享POL III加工的能力。这些实验的结果将为DNA复制，DNA修复和TLS的坐标调节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的过程中的关键步骤，这些步骤可以针对控制能力和复制的忠诚度以获得治疗性增益。