Using DNA Curtains to Reveal the Mechanisms of Target Site Location by DNA Binding Proteins

智力价值：该项目解决了生物学中的一个基本问题：特定部位和结构的DNA结合蛋白如何在大量的非特定DNA中定位其目标？这个问题将采用涵盖纳米工程、表面化学、物理和生物化学的多学科方法进行研究。蛋白质定位稀有靶点的能力对基因表达、DNA复制、染色体动力学和基因组维持的各个方面都是必不可少的，但这些过程如何发生的有意义的细节通常是无法获得的。该项目将专注于复制后错配修复(MMR)作为一个模型系统，并将进行实验，以准确确定MMR蛋白MUTSA和MUTRA如何定位和响应其特定的DNA靶标。尽管进行了多年的深入研究，但这些过程的详细机制尚不清楚，这主要是由于总体水平的生化测量的固有限制。为了克服这些限制，该项目将利用纳米级的“DNA窗帘”，并应用全内反射荧光显微镜(TIRFM)来直接显示单个蛋白质复合体，因为它们在DNA的单个分子上搜索和接触他们的目标位置。这种独特的单分子成像方法由PI的实验室首创，通过对多个反应轨迹进行并行成像，实现了从单个分子实时快速收集统计相关信息。更广泛的影响-格林博士的实验室开发的技术为实时、单分子的核蛋白复合体分析提供了一种高通量方法。PI积极地将这项技术传播给更广泛的研究社区，因为它有可能为涉及蛋白质和DNA分子之间相互作用的几乎任何生物系统提供新的见解。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从事重要的科学职业。为了促进对生物科学单分子方法的理解，这些技术已被整合到大学的本科生和研究生课程中。国际学生联合会致力于共同努力，推动年轻学生的科学培训和发展，特别是通过以研究为导向的教育计划。他是哥伦比亚大学夏季本科生研究奖学金项目(SURF)的本科生导师，也是哈莱姆儿童协会的学生导师。该项目将把较年轻的学生融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。

Intellectual Merit: The project addresses a fundamental problem in biology: How do site- and structure-specific DNA-binding proteins locate their targets among a vast excess of nonspecific DNA? This question will be studied using a multi-disciplinary approach encompassing nanoscale engineering, surface chemistry, physics and biochemistry. The ability of proteins to locate rare targets is essential for all aspects of gene expression, DNA replication, chromosome dynamics and genome maintenance, yet meaningful details of how these processes occur are typically unavailable. This project will focus on post-replicative mismatch repair (MMR) as a model system, and experiments will be conducted to determine precisely how the MMR proteins MutSa and MutLa locate and respond to their specific DNA targets. Detailed mechanisms of these processes are not yet available, despite years of intensive investigation, largely due to the inherent limitations of ensemble-level biochemical measurements. To overcome these limitations, this project will utilize nanofabricated' DNA Curtains' and apply total internal reflection fluorescence microscopy (TIRFM) to directly visualize individual protein complexes as they search for and engage their target sites on individual molecules of DNA. This unique approach to single-molecule imaging was pioneered by the PI's laboratory, and enables rapid collection of statistically relevant information in real time from individual molecules by enabling parallel imaging of multiple reaction trajectories. Broader Impacts - The technology developed in Dr. Greene's laboratory provides a 'high-throughput' approach for real-time, single-molecule analysis of nucleoprotein complexes. The PI has actively disseminated this technology to the broader research community, as it has the potential to provide novel insights into virtually any biological system that involves 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 pursue significant scientific careers upon completion of their degree requirements. To promote the understanding of single-molecule approaches to biological science, these technologies have been integrated into the University's undergraduate and graduate course curriculum. The PI is engaged in concerted efforts to advance the scientific training and development of younger students, particularly through research-oriented educational programs. He serves as an undergraduate student mentor for Columbia University's Summer Undergraduate Research Fellowship program (SURF), and as a student mentor for the Harlem Children's Society. The project will integrate younger students into all aspects of scientific work performed in the laboratory, thereby providing them with valuable, real-world research experiences.