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: 8288098
• 负责人: MARK A AKESON
• 金额: $ 26.9万
• 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/8288098
• 关键词: B-DNA; Bacteriophage T7; Binding; Biochemical; Biophysics; Cancer Etiology; Catalysis; Complex; Coupled; Coupling; DNA; DNA Polymerase I; DNA biosynthesis; DNA-Directed DNA Polymerase; Detection; Discrimination; Electronics; Environmental Impact; Enzymes; Escherichia coli; Exhibits; 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）： 项目摘要 该应用旨在在与酶结合和催化速率叠加的时间尺度上实现 DNA 聚合酶功能的电子控制。为了实现这一目标，我们将监测电压诱导张力下单个 DNA 聚合酶与纳米孔传感器的相互作用。我们将表征纳米孔中电压控制下捕获的聚合酶-DNA 复合物的动力学、生化和结构特性。我们将以比使用传统技术更高的带宽优化聚合酶功能的纳米孔测量，并允许在处理 DNA 时对数千种单独的酶进行连续分析。我们相信这项研究是创新的，因为它将采用最近建立的纳米孔技术来识别和测量单个催化复制循环期间的易位步骤。聚合酶驱动的易位机制之间的区分应该是可以实现的。这项工作与人类健康相关，因为 DNA 聚合酶错误掺入核苷酸而产生的突变是癌症的根本原因。此外，纳米孔偶联聚合酶可以为基因组测序提供一种高速、低成本的技术，而且几乎对环境没有影响。 公共卫生相关性： 这项工作的重点是 DNA 聚合酶进行 DNA 复制的机制。它与人类健康相关，因为 DNA 聚合酶错误掺入核苷酸而产生的突变是癌症的根本原因。此外，纳米孔偶联聚合酶可以为基因组测序提供一种高速、低成本的技术，而且几乎对环境没有影响。