CAREER: Using High-throughput Single-molecule Analysis to Reveal the Mechanisms of Target Site Location by DNA Repair Proteins

职业：利用高通量单分子分析揭示 DNA 修复蛋白的靶位点定位机制

• 批准号: 0544638
• 负责人: Eric Greene
• 金额: $ 90.21万
• 依托单位: Columbia University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-15 至 2012-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0544638&HistoricalAwards=false
• 关键词: CAREER; Using; throughput; Single; molecule

This research incorporates micro-scale materials engineering, surface chemistry, physics and biochemistry to answer fundamental questions about biology that cannot easily be addressed through traditional experimental methods. The overall goal encompasses a classic problem in biochemistry, namely: How do site- or structure-specific DNA-binding proteins locate their targets among a vast excess of nonspecific DNA? To help address this question, the Greene laboratory is using total internal reflection fluorescence microscopy (TIRFM) as a tool to directly visualize individual protein complexes as they search for their target sites on single molecules of DNA. The Greene laboratory is also developing new methods that will allow the construction of aligned arrays comprised of hundreds of individual DNA molecules, which are suspended above an inert lipid bilayer and organized into patterns with user-defined orientations, tensions, and topologies. These DNA arrays will allow for rapid collection of statistically relevant information from hundreds of individual molecules by making possible parallel processing of multiple reaction trajectories. These novel research tools will be used to determine how proteins that are involved in post-replicative repair of mismatched bases locate and respond to their specific targets. Despite years of intensive investigation these mechanisms remain unknown, largely due to the inherent limitations of traditional ensemble-level biochemical measurements. These new single-molecule approaches can be used to determine exactly what proteins are bound to DNA, where they are bound, how they behave, when they leave, and how they influence one another, all in real-time at the single-molecule level.The technology-driven methods developed during the course of this research will provide a high-throughput approach for single-molecule analysis of nucleoprotein complexes, which can be applied towards the study of virtually any biological system that involves the interactions between protein and DNA molecules. This interdisciplinary work also provides trainees with a cutting-edge, broad-based educational experience that will allow them to successfully contribute to the scientific community upon completion of their degree requirements. To promote the understanding of single-molecule approaches, these emerging technologies will be integrated into the university's graduate course curriculum; several departmental lectures will be scheduled featuring leading experts from around the country; and a regional discussion group/symposium will also be organized to stimulate interactions and communication between the laboratories in the New York area that are interested in single-molecule research. Dr. Greene has initiated a separate project that will be conducted solely by undergraduate and high school students. The goal of these efforts is to incorporate younger students into all aspects of scientific work performed in the laboratory, thereby providing them with valuable, real-world research experience.

这项研究结合了微观材料工程、表面化学、物理学和生物化学，以回答传统实验方法无法轻易解决的生物学基本问题。总体目标涵盖了生物化学中的一个经典问题，即：位点或结构特异性 DNA 结合蛋白如何在大量非特异性 DNA 中定位其靶标？为了帮助解决这个问题，Greene 实验室正在使用全内反射荧光显微镜 (TIRFM) 作为工具，在单个蛋白质复合物在单分子 DNA 上寻找目标位点时直接可视化单个蛋白质复合物。 Greene 实验室还在开发新方法，该方法将允许构建由数百个单独 DNA 分子组成的对齐阵列，这些分子悬浮在惰性脂质双层上方，并组织成具有用户定义的方向、张力和拓扑的图案。这些 DNA 阵列可以通过并行处理多个反应轨迹，从而快速收集数百个单个分子的统计相关信息。这些新颖的研究工具将用于确定参与错配碱基复制后修复的蛋白质如何定位和响应其特定靶点。尽管经过多年的深入研究，这些机制仍然未知，这很大程度上是由于传统整体水平生化测量的固有局限性。这些新的单分子方法可用于准确确定哪些蛋白质与 DNA 结合、它们结合的位置、它们的行为方式、何时离开以及它们如何相互影响，所有这些都在单分子水平上实时进行本研究过程中开发的技术驱动方法将为核蛋白复合物的单分子分析提供高通量方法，该方法可应用于几乎任何涉及蛋白质和 DNA 分子之间相互作用的生物系统的研究。这项跨学科工作还为学员提供了前沿、基础广泛的教育经验，使他们能够在完成学位要求后成功为科学界做出贡献。为了促进对单分子方法的理解，这些新兴技术将被纳入大学的研究生课程；将安排一些院系讲座，邀请来自全国各地的顶尖专家参加；还将组织区域讨论组/研讨会，以促进纽约地区对单分子研究感兴趣的实验室之间的互动和交流。格林博士启动了一个单独的项目，该项目将仅由本科生和高中生进行。这些努力的目标是将年轻学生纳入实验室进行的科学工作的各个方面，从而为他们提供宝贵的、真实的研究经验。