Study of the Dynamics of Protein-DNA Interactions to Probe Site-Specific Recognition

University Of Illinois At Chicago, Chicago IL

Investigators

Abstract

The overall goals of this study are to probe the dynamics of molecular rearrangements in both the protein and the DNA during complex formation and to provide a physical basis for elucidating the molecular origins of sequence- and structure-specificity. This project will focus on two classes of proteins: (i) three closely related eubacterial DNA-bending proteins involved in DNA packaging and gene regulation that dramatically bend the DNA, by nearly 180 degrees: E. coli Integration Host Factor (IHF), histone-like protein from Anabeana (AHU), and Hbb from the Lyme-disease causing spirochete Borrelia burgdorferi; and (ii) a DNA-repair protein, MutS, that recognizes and binds to DNA sites with a mismatch, and bends the DNA by about 60 degrees, thus initiating the DNA repair machinery. These proteins recognize their binding sites primarily by an indirect readout mechanism, in which the sequence-dependent structure and flexibility/bendablity of the DNA play a key role. A ~10 nanosecond laser temperature-jump (T-jump) will be used to perturb the protein-DNA complex, and the bending/unbending dynamics of the bound DNA substrate will be monitored with time-resolved FRET on end-labeled DNA substrates. In addition, protein conformational changes in response to the T-jump will be monitored with Trp fluorescence changes of intrinsic or introduced Trp residues. Single-molecule FRET measurements will also be carried out on immobilized DNA substrates with bound protein to probe the distribution of bent conformations in the complex, and to provide dynamics information at the single-molecule level. The specific aims of this study are to (i) probe the role of DNA flexibility/bendability in the recognition mechanism by measuring the kinetics of DNA bending/unbending for a range of substrates with inserted distortions such as mismatches or single-T insertions that bind with widely varying affinities to the proteins in the IHF/HU family; (ii) probe directly the conformational changes in the protein to address the question: do protein conformational changes occur concurrently with the DNA bending/unbending step or in a distinct kinetic step; (iii) probe the nature of the transition state along the reaction coordinate for complex formation by investigating the effect of mutations that perturb specific protein-DNA interactions on the DNA bending/unbending rates; (iv) probe the DNA bending kinetics in mismatched substrates bound to MutS to investigate how DNA bending dynamics influence mismatch recognition by MutS and subsequent ATPase-driven steps in the DNA repair mechanism. A novel aspect of this project is the application of laser T-jump techniques to probe the dynamics of protein-DNA interactions with submicrosecond time-resolution. The broader impact of this work is in the potential for extending these kinetics measurements to a wider class of protein-DNA systems, including other regulatory and DNA-repair proteins, for a deeper understanding of the underlying mechanisms. The primary educational goal is to establish an undergraduate Biophysics major at UIC, which will provide a multidisciplinary education with a strong analytical component. This project is being jointly supported by Molecular Biophysics in the Division of Molecular and Cellular Biosciences and the Biological Physics Program in the Physics Division.