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

利用 DNA 窗帘揭示 DNA 结合蛋白的靶位点定位机制

• 批准号: 1154511
• 负责人: Eric Greene
• 金额: $ 112.5万
• 依托单位: Columbia University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1154511&HistoricalAwards=false
• 关键词: Using; DNA; Curtains; Reveal; Mechanisms

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.

智力优点：该项目解决了生物学中的一个基本问题：位点和结构特异性 DNA 结合蛋白如何在大量非特异性 DNA 中定位其目标？该问题将采用涵盖纳米工程、表面化学、物理学和生物化学的多学科方法进行研究。蛋白质定位稀有靶标的能力对于基因表达、DNA 复制、染色体动力学和基因组维护的各个方面都至关重要，但这些过程如何发生的有意义的细节通常无法获得。该项目将重点关注复制后错配修复 (MMR) 作为模型系统，并将进行实验以确定 MMR 蛋白 MutSa 和 MutLa 如何定位和响应其特定 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，这主要是由于整体水平生化测量的固有局限性。为了克服这些限制，该项目将利用纳米制造的“DNA 窗帘”并应用全内反射荧光显微镜 (TIRFM)，在单个蛋白质复合物搜索并接合单个 DNA 分子上的目标位点时直接可视化单个蛋白质复合物。这种独特的单分子成像方法是由 PI 实验室首创的，通过对多个反应轨迹进行并行成像，可以快速从单个分子中实时快速收集统计相关信息。更广泛的影响 - Greene 博士实验室开发的技术为核蛋白复合物的实时单分子分析提供了一种“高通量”方法。 PI 积极向更广泛的研究界传播这项技术，因为它有潜力为几乎所有涉及蛋白质和 DNA 分子之间相互作用的生物系统提供新的见解。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，这些技术已被纳入大学的本科生和研究生课程中。 PI 致力于促进年轻学生的科学培训和发展，特别是通过研究型教育项目。他担任哥伦比亚大学夏季本科生研究奖学金计划 (SURF) 的本科生导师，以及哈莱姆儿童协会的学生导师。该项目将让年轻学生融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。