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.