Analysis of Single DNA Polymerase Complexes at 5 Angstrom Precision in Real Time

以 5 埃精度实时分析单个 DNA 聚合酶复合物

• 批准号: 7980777
• 负责人: MARK A AKESON
• 金额: $ 27.9万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7980777
• 关键词: B-DNA; Bacteriophage T7; Binding; Biochemical; Biophysics; Cancer Etiology; Catalysis; Complex; Coupling; DNA; DNA Polymerase I; DNA biosynthesis; DNA-Directed DNA Polymerase; Detection; Discrimination; Electronics; Environmental Impact; Enzymes; Escherichia coli; Exhibits; Figs - dietary; Genetic Transcription; Genetic Translation; Genome; Goals; Health; Health Sciences; Human; Individual; Kinetics; Measurement; Measures; Modeling; Monitor; Movement; Mutation; Nucleotides; Pathway interactions; Polymerase; Process; Property; Publications; RNA; RNA analysis; Resolution; Specificity; Speed; Structure; Techniques; Technology; Testing; Time; Translations; Work; base; cost; cost effective; electric field; experience; genome sequencing; improved; innovation; instrument; nanopore; nanoscale; public health relevance; sensor; single cell analysis; single molecule; voltage

DESCRIPTION (provided by applicant): Project Summary This application aims to achieve electronic control of DNA polymerase function on a time scale that superimposes with rates of enzyme binding and catalysis. To achieve this aim, we will monitor the interaction of individual DNA polymerases with a nanopore sensor under voltage- induced tension. We will characterize kinetic, biochemical, and structural properties of polymerase-DNA complexes captured under voltage control in a nanopore. We will optimize nanopore measurements of polymerase function at significantly higher bandwidth than is possible using conventional techniques and in a manner that permits serial analysis of thousands of individual enzymes as they process DNA. We believe the study is innovative because it will employ a recently established nanopore technique to identify and measure translocation steps during individual catalytic cycles of replication. Discrimination between polymerase-driven translocation mechanisms should be achievable. This work is relevant to human health because mutations that arise from misincorporation of nucleotides by DNA polymerases are a fundamental cause of cancer. In addition, nanopore-coupled polymerases could present a high speed, low cost technology for genome sequencing that has virtually no environmental impact. PUBLIC HEALTH RELEVANCE: This work focuses on mechanisms of DNA replication by DNA polymerases. It is relevant to human health because mutations that arise from misincorporation of nucleotides by DNA polymerases are a fundamental cause of cancer. In addition, nanopore-coupled polymerases could present a high speed, low cost technology for genome sequencing that has virtually no environmental impact.

描述（由申请人提供）：