Dynamics and Mechanism of DNA-Bending Proteins in Binding Site Recognition

许多细胞过程涉及蛋白质和DNA之间的相互作用，其中蛋白质识别并结合DNA上的特定位点，其亲和力比随机DNA序列高数千倍或数百万倍。这些蛋白质如何在大量的非特异性位点中搜索并找到它们的特异性位点仍然是一个谜。许多位点特异性蛋白质在该位点扭结、弯曲或扭曲DNA，并利用序列依赖性DNA变形性来识别它们的结合位点（间接读出），并经历构象重排以促进与弯曲DNA的有利相互作用（诱导配合）。一个关键问题仍然存在：蛋白质是否使DNA弯曲（蛋白质诱导的弯曲），或者是在不存在蛋白质的情况下DNA热可接近的部分弯曲构象，蛋白质捕获该构象以形成紧密复合物（构象捕获）。为了阐明这两种机制之间的相互作用，需要测量DNA的构象分布，有和没有结合的蛋白质，和蛋白质和DNA的构象变化的动力学测量沿着从非特异性到特异性复合物的过渡途径。蛋白质-DNA复合物的大部分动力学测量都来自停流测量，这种测量无法捕获DNA弯曲动力学，甚至最基本的问题也没有得到回答：复合物中的DNA弯曲在什么时间尺度上发生。该项目的一个新的方面是激光温度跳跃技术的应用，以延长动力学测量的时间分辨率为亚微秒，其中包括相关的识别步骤的时间尺度。结合其他方法，如单分子FRET和皮秒分辨的荧光衰减测量，这项研究将产生难以捉摸的DNA弯曲步骤和构象状态的分布访问DNA，提供一个全面的了解相对贡献的构象捕获与蛋白质诱导的弯曲。该研究将集中在两类DNA弯曲蛋白：MutS，识别DNA中的错配并启动修复机制; EcoRV，一种限制性酶，识别外源DNA上的特定靶序列并以高特异性切割它。主要目标是：研究同源（特异性）DNA序列是否具有在不存在结合蛋白的情况下采用弯曲构象的内在倾向;确定序列依赖性柔性/变形性是否影响复合物中DNA弯曲的速率;并阐明导致结合位点识别的分子重排序列。长期目标是将这些测量扩展到识别不同类型DNA损伤的蛋白质，包括化学修饰的核苷酸，其他损伤修复系统，以及参与基因调控的其他DNA弯曲蛋白质，以统一理解内在DNA力学和识别机制中的灵活性。从这项研究中获得的见解将对理解蛋白质-DNA相互作用中间接读出的基本规则产生影响。该项目将有助于本科生，研究生和博士后学生的专业培训，通过他们参与研究，使用最先进的生物物理方法，旨在揭开难以捉摸的蛋白质-DNA动力学。研究的各个方面将在生物学和物理学的界面上融入课堂教学。实验将为本科生设计，基于单分子荧光仪器，这是该项目的一个组成部分，提供动手接触现代生物学中的重要概念，如扩散，波动和相关性测量，通常不包括在生物学或物理课程。一个新的生物物理学入门课程，将整合这些和其他主题，将开发，以增加以前开发的高级分子生物物理学课程。

Many cellular processes involve interactions between proteins and DNA in which proteins recognize and bind to specific sites on the DNA with thousand- or million-fold higher affinities than to random DNA sequences. How these proteins search and find their specific sites in the midst of a large excess of nonspecific sites remains a puzzle. Many site-specific proteins kink, bend or twist DNA at that site, and exploit the sequence-dependent DNA deformability to recognize their binding site (indirect readout), and undergo conformational rearrangements to facilitate favorable interactions with the bent DNA (induced-fit). A key question remains: does the protein bend the DNA (protein-induced bending) or are partially bent conformations thermally accessible to DNA in the absence of the protein, which the protein captures to form a tight complex (conformational capture). To elucidate the interplay between these two mechanisms requires measurements of the conformational distribution of DNA, with and without bound protein, and kinetics measurements of conformational changes in protein and DNA along the transition pathway from nonspecific to specific complex. The bulk of kinetics measurements on protein-DNA complexes have come from stopped-flow measurements that were unable to capture DNA-bending kinetics, leaving unanswered even the most basic question: on what time scales does the DNA bend in the complex. A novel aspect of this project is the application of laser temperature-jump techniques to extend the time resolution for kinetics measurements to submicroseconds, which covers the time scale relevant for the recognition step. In combination with other approaches such as single-molecule FRET and picoseconds-resolved fluorescence decay measurements, this study will yield the elusive DNA bending step and the distribution of conformational states accessible to DNA, to provide a comprehensive understanding of the relative contributions of conformational capture versus protein-induced bending. The study will focus on two classes of DNA-bending proteins: MutS, that recognizes mismatches in DNA and initiates the repair machinery; and EcoRV, a restriction enzyme that recognizes a specific target sequence on foreign DNA and cleaves it with high specificity. The primary goals are: to investigate whether the cognate (specific) DNA sequences have an intrinsic propensity to adopt bent conformations in the absence of bound protein; to determine whether sequence-dependent flexibility/deformability influences the rate at which DNA is bent in the complex; and to elucidate the sequence of molecular rearrangements that lead to binding site recognition. The long term goals are to extend these measurements to proteins that recognize different kinds of DNA damages, including chemically modified nucleotides, other damage repair systems, as well as other DNA-bending proteins involved in gene regulation, for a unified understanding of the role of intrinsic DNA mechanics and flexibility in the recognition mechanism. The insights gained from this study will have an impact on understanding the fundamental rules that govern indirect readout in protein-DNA interactions. This project will contribute to the professional training of undergraduates, graduates, and postdoctoral students by their involvement in research, using state-of-the-art biophysical approaches designed to unveil elusive protein-DNA dynamics. Aspects of the research will be integrated in classroom teaching at the interface of biology and physics. Experiments will be designed for undergraduates, based on the single-molecule fluorescence apparatus, which is an integral part of this project, to provide hands-on exposure to important concepts in modern biology such as diffusion, fluctuations, and correlation measurements that are typically not covered in biology or physics curricula. A new introductory biophysics course, which will integrate these and other topics, will be developed to augment a previously developed upper-level molecular biophysics course.