Thermodynamics and kinetics of DNA binding protein probed by DNA overstretching

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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.