Probing Conformational Transition Pathways of Protein-DNA Complexes by Polarization-Modulated Fourier Imaging Correlation Spectroscopy (PM-FICS)

生命过程化学（CLP）项目的这一奖项支持俄勒冈大学的Andrew H. Marcus教授对DNA聚合酶和解旋酶分子机制的基础研究，这两种蛋白质-DNA复合物是制定和调节基因表达的生物过程的基本要素。在双链和引物模板DNA的分叉和连接处，对荧光核酸碱基类似物取代了天然核苷酸。裸DNA结构的偏振荧光波动测量将揭示碱基模拟位点的相对偶极子角及其在微秒至亚秒时间尺度上的相对运动信息。后续的实验将在蛋白质- dna复合物上进行，以直接探测在复制过程的关键步骤中蛋白质酶位点的机制。这些研究将提供有关DNA合成过渡状态途径的详细信息，并研究可能存在的与复制保真度有关的中间状态。这项研究的一个重要组成部分是积极招募和指导来自代表性不足地区的学生参加本科和研究生培训项目。这些研究将增加我们对动态过程的基本理解，即基因表达的大分子机器在执行其生物学功能时运作。潜在的好处包括提高对化学和生物行为之间分子水平界面的理解，以及对纳米机器基本原理的新见解。

This award in the Chemistry of Life Processes (CLP) program supports work by Professor Andrew H. Marcus at the University of Oregon to carry out fundamental studies on the molecular mechanisms of DNA polymerase and helicase, two protein-DNA complexes that are essential elements of the biological processes that enact and regulate gene expression. Pairs of fluorescent nucleic acid base analogues are substituted for native nucleotides at the forks and junctions of double stranded and primer-template DNA. Polarized fluorescence fluctuation measurements of the naked DNA constructs will reveal information about the relative dipole angle of the base analogue sites, and their relative motions on microsecond to sub-second time scales. Subsequent experiments will be carried out on protein-DNA complexes to directly probe the mechanics of the protein enzymatic sites during key steps of the replication process. These studies will provide detailed information about the transition state pathways of DNA synthesis, and investigate the possible existence of intermediate states involved in replication fidelity. A significant component of the research involves active recruitment and mentoring of students from under-represented areas into the undergraduate and graduate training programs. These studies will add to our basic understanding of the dynamic processes whereby the macromolecular machines of gene expression operate in performing their biological functions. Potential benefits range from an improved understanding of the molecular level interface between chemical and biological behavior, to new general insights about the underlying principles of nano-machines.