CAREER: Investigation of DNA-binding Protein Dynamics With High-resolution Optical Traps

一类广泛的dna结合蛋白以非序列特异性的方式与基因组相互作用。这些蛋白质在它们的底物上机械地起作用，通过弯曲、扭曲或拉伸分子来改变DNA的构象，并通过寡聚形成长丝状结构。这些核蛋白复合物通常作为基因组维持过程发生的底物。因此，它们参与DNA代谢、复制、重组和修复的各个方面，是细胞过程的重要调节因子。单链DNA结合蛋白（SSB）作为这类蛋白共同特征的模型系统。该项目将使用传统生物化学和分子生物学的综合技术，结合单分子生物物理学和计算生物学来研究：(1)SSBs如何诱导核酸的构象重排，(2)它们如何寡聚成核蛋白细丝，以及(3)这些蛋白质簇如何招募和调节参与核酸加工的其他蛋白质的活性。具体来说，高分辨率光学捕获结合单分子荧光技术将用于揭示动态蛋白质- dna相互作用，超越当前方法的局限性。这项工作将阐明基因组维持的基本方面。更广泛的影响：PI致力于青年科学家的跨学科教育，并向代表性不足的群体伸出援手。特别是，他认为生物物理学是一个独特的机会，可以招募传统上更喜欢生物学而不是物理学的女性进入定量科学领域。项目的推广和教育部分将这些主题综合成一个针对初中和高中、本科和研究生教育的广泛计划。具体而言，PI将：(1)为女生暑期项目开设实验室营，向初高中女生讲授物理学及其对生物问题的影响，并提供生物物理学的实践经验；(2)改进面向生命科学专业学生（其中大多数是女性）的本科物理入门课程的教学，以更好地将物理概念与生物学和医学联系起来；(3)作为美国国家科学基金会活细胞物理中心（CPLC）的一部分，开发一门致力于培养下一代先进技术生物物理学家的实验课；(4)参加年度少数人会议。本项目由分子与细胞生物科学部的基因与基因组系统组和生物分子系统组共同支持。

A broad class of DNA-binding protein interacts with the genome in a non-sequence-specific manner. These proteins act mechanically on their substrates, altering DNA conformation by bending, twisting, or stretching the molecule, and oligomerizing to form long filaments. These nucleoprotein complexes often serve as substrates upon which genome maintenance processes occur. Thus, they are involved in all aspects of DNA metabolism, in replication, recombination, and repair, and are important regulators of cellular processes. Single-stranded DNA binding proteins (SSB) serve as a model system for features common to this class of proteins. This project will use a synthesis of techniques from traditional biochemistry and molecular biology, in combination with single-molecule biophysics and computational biology to investigate: (1) how SSBs induce conformational rearrangement of nucleic acids, (2) how they oligomerize into nucleoprotein filaments, and (3) how these protein clusters recruit and modulate the activity of other proteins involved in nucleic acid processing. Specifically, high-resolution optical trapping in combination with single-molecule fluorescence techniques will be used to reveal dynamic protein-DNA interactions, going beyond the limitations of current methods. This work will shed light on fundamental aspects of genome maintenance.Broader impact: The PI has a deep commitment to interdisciplinary education of young scientists and outreach towards underrepresented groups. In particular, he sees in biophysics a unique opportunity to recruit women, who have traditionally been drawn to biology over physics, into the quantitative sciences. The outreach and education components of the project synthesize these themes into a broad plan targeting middle and high school, undergraduate, and graduate education. Specifically, the PI will: (1) develop a lab camp for a girls' summer program to teach middle and high school girls about physics and its impact on biological problems and to provide hands-on experience with biophysics, (2) improve the teaching of an introductory undergraduate physics course for life science students (the majority of whom are women) to better connect physics concepts with biology and medicine, (3) develop a lab course devoted to the training of the next generation of biophysicists in advanced technologies as part of the NSF Center for the Physics of the Living Cells (CPLC), and (4) participate in yearly minority conferences. This project is jointly supported by the Genes and Genome Systems Cluster and the Biomolecular Systems Cluster in the Division of Molecular and Cellular Biosciences.