Thermodynamics and kinetics of DNA binding protein probed by DNA overstretching

描述（由申请人提供）：DNA的结构转变和蛋白质在DNA上的对接是分子生物学的基本过程。了解和控制这些过程对于设计抗传染病（如艾滋病毒）的新药至关重要。为了更好地理解DNA复制的生物学过程，研究细胞中发生的不同条件下单个DNA分子和与DNA相互作用的分子的性质是很重要的。单分子力谱已被证明是一种研究DNA二级结构变化的通用技术；然而，这项技术的应用仍处于起步阶段。目前，单双链DNA分子被光学镊子仪器拉伸，在约65 pN的拉伸力下，可以诱导DNA双链的两条链解绕（螺旋-线圈转变）。通过牵引产生的解绕DNA的局部区域与单链DNA结合蛋白的相互作用可以在广泛的条件下进行探测。这些新的实验技术是由最近发展的先进的生物物理和螺旋-线圈过渡计算模型补充的。具体来说，噬菌体T7基因2.5蛋白（gp2.5）是一种单链DNA结合蛋白，在噬菌体感染细胞的DNA复制和重组中起重要作用。通过研究gp2.5存在下速率依赖的DNA融化力，以蛋白质结合速率和解离速率以及平衡结合常数来测量蛋白质与单链DNA结合的动力学和热力学。该测量的一个重要参数是DNA中螺旋线圈边界的数量在过拉伸过渡时作为测量时间的函数。

DESCRIPTION (provided by applicant): Structural transitions of DNA and docking of proteins on DNA are fundamental processes in molecular biology. Understanding and controlling these processes are essential to the design of novel drugs against infectious diseases (e.g., HIV). In order to better understand the biological process of DNA replication, it is important to study the properties of single DNA molecules and molecules that interact with DNA under different conditions occurring in the cell. Single-molecule force spectroscopy has proved to be a versatile technique for investigating changes in DNA secondary structure; however, applications of this technique are still in their infancy. Currently, single duplex DNA molecules are stretched by an optical tweezers instrument, which at a stretching force of about 65 pN can induce the unwinding of the two strands of the DNA duplex (helix-coil transition). The interaction of the localized regions of unwound DNA generated by traction with single-stranded DNA binding proteins can then be probed under a wide range of conditions. These new experimental techniques are complemented by the recent development of advanced biophysical and computational models for the helix-coil transition. Specifically, Bacteriophage T7 gene 2.5 protein (gp2.5) is a single-stranded DNA binding protein that has essential roles in DNA replication and recombination in phage-infected cells. By studying the rate-dependent DNA melting force in the presence of gp2.5, the kinetics and thermodynamics of protein binding to single-stranded DNA is measured in terms of protein association and dissociation rates and the equilibrium association constant. An important parameter for this measurement is the number of helix-coil boundaries in the DNA at the overstretching transition as a function of measurement time. The main objective of this research is to develop a comprehensive biophysical model for stretched DNA and its interaction with single-stranded DNA binding proteins. Specifically, we will use this model to determine the experimental parameter mentioned above. The results of this project are expected to shed new light on fundamental biological processes of infectious diseases. This project is the continuation of a S06 SCORE pilot project with the same general scope. It is a starting point for future long-term collaborative research and will therefore be an important tool for the PI's faculty development.