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

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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.