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

这项研究结合了微尺度材料工程，表面化学，物理学和生物化学，以回答无法通过传统实验方法轻松解决的生物学基本问题。总体目标包含了生物化学中的一个经典问题，即：位点特异性或结构特异性DNA结合蛋白如何在大量过量的非特异性DNA中定位其靶点？为了帮助解决这个问题，格林实验室正在使用全内反射荧光显微镜（TIRFM）作为一种工具，直接可视化单个蛋白质复合物，因为它们在单个DNA分子上寻找它们的靶位点。格林实验室还在开发新的方法，这些方法将允许构建由数百个单独的DNA分子组成的对齐阵列，这些DNA分子悬浮在惰性脂质双层上，并组织成具有用户定义的方向，张力和拓扑结构的模式。这些DNA阵列将允许从数百个单个分子中快速收集统计相关信息，从而使多个反应轨迹的并行处理成为可能。这些新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标。尽管多年的深入研究，这些机制仍然未知，主要是由于传统的整体水平的生化测量的固有局限性。这些新的单分子方法可用于确定哪些蛋白质与DNA结合，它们在哪里结合，它们如何行为，何时离开，以及它们如何相互影响，所有这些都是在单分子水平上实时进行的。在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，它可以应用于研究几乎任何涉及蛋白质和DNA分子之间相互作用的生物系统。这项跨学科的工作还为学员提供了一个前沿的，基础广泛的教育经验，这将使他们能够成功地为科学界作出贡献后，完成他们的学位要求。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;将安排几个部门讲座，由来自全国各地的顶尖专家主讲;还将组织一个区域讨论组/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。格林博士发起了一个单独的项目，将由本科生和高中生单独进行。这些努力的目标是将年轻的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，真实世界的研究经验。

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.