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

这项研究融合了微尺度材料工程、表面化学、物理和生物化学，以回答有关生物学的基本问题，这些问题无法通过传统的实验方法轻松解决。总的目标围绕着生物化学中的一个经典问题，即：位置或结构特定的DNA结合蛋白如何在大量的非特定DNA中定位其靶标？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，在单个蛋白质复合体搜索其在DNA单分子上的目标位置时直接显示它们。格林实验室还在开发新的方法，可以构建由数百个单独的DNA分子组成的排列阵列，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阵列将使并行处理多个反应轨迹成为可能，从而允许从数百个单独的分子中快速收集统计上相关的信息。这些新的研究工具将被用来确定参与错配碱基复制后修复的蛋白质如何定位并对其特定目标做出反应。尽管进行了多年的深入研究，但这些机制仍不清楚，这主要是由于传统的系综水平生化测量的内在局限性。这些新的单分子方法可以用来准确地确定什么蛋白质与DNA结合，它们在哪里结合，它们如何行为，它们何时离开，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。在这项研究过程中开发的技术驱动的方法将为核蛋白复合体的单分子分析提供一种高通量的方法，可以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。这项跨学科的工作还为学员提供了前沿的、基础广泛的教育经验，使他们能够在完成学位要求后成功地为科学界做出贡献。为了促进对单分子方法的理解，这些新兴技术将被整合到大学的研究生课程中；将安排几场有来自全国各地的顶尖专家参加的系级讲座；还将组织一个区域讨论小组/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，该项目将只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方方面面，从而为他们提供宝贵的现实世界研究经验。

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.