Protein Diffusion and Interaction in Concentrated DNA Solutions

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9728165 Bloomfield DNA functions in crowded, strongly interacting solutions, mainly while interacting with proteins. The equilibrium and dynamic aspects of protein-DNA interactions are strongly influenced by the environment. When the volume occupancy of a solution reaches more than thirty percent, as it does within cells, simple extrapolations from dilute solution behavior become totally inadequate. The goal in this project is to develop a better experimental and theoretical understanding of the ways in which high concentrations of DNA and other polymers influence the diffusional dynamics of proteins which bind to DNA. The three specific aim are (1) To characterize the diffusional motion of proteins within a crowded DNA environment and in DNA solutions which also contain crowding agents such as dextran, Ficoll, and PEG, so as to understand the effects of excluded volume, ionic interactions, and specific binding interactions. (2) To determine how crowding influences the relative importance of one-dimensional and three-dimensional diffusion in the action of restriction enzymes. (3) To use Brownian dynamics simulation of protein diffusion in crowded DNA solutions, to gain insight into the ways in which ionic strength-dependent volume exclusion and nonspecific binding affect the search of regulatory proteins for their specific binding sites. The major tools will be fluorescence photobleaching recovery and dynamic light scattering to measure diffusion, gel electrophoresis to follow kinetics of restriction enzyme action, and Brownian dynamics to simulate diffusion in complex, interacting systems. The general significance of this research is to gain insight into the effect of the crowded, strongly interacting conditions prevailing inside cells, and how these conditions affect the dynamic diffusion and reaction processes involved in gene regulation. ***