9410748 Brenowitz Photochemical techniques have been researched and tested enabling time-resolved determinations of the structures of protein-DNA complexes that allow the reaction intermediates to be examined. This project will 1) Determine the mechanism and specificity of cleavage of DNA by photolyzed cobalamins; 2) Develop a rapid mixing-laser photolysis method to attain the goal of millisecond time resolution for photochemical footprinting; and 3) Test the new technology by examining the reaction mechanism of two sequence- specific DNA binding proteins. This new technique will be immensely valuable in bridging the gaps in present thermodynamic and structural analysis of protein-DNA interactions. Since complex formation can be followed at single basepair resolution, detailed structural information will be available at time resolutions on the order of milliseconds to microseconds. The photochemical footprinting protocols will be tested using the binding of the Eco RI restriction endonuclease to its recognition sequence and the binding of the 'TATA' Binding Protein (TBP) to a promoter. Thus, it will be possible to study for the first time the intermediate steps in the formation of the protein-nucleic acid complex. %%% Understanding the mechanisms by which proteins recognize and bind to specific DNA sequences is a key step towards understanding cellular function and dysfunction. Protein-DNA interactions play important roles in cellular processes such as gene replication, recombination and regulation. The formation of protein DNA complexes is a complex process involving multiple steps and many different chemical interactions. Significant insights have been made into the mechanisms by which proteins bind to specific DNA sequences using techniques that determine the three-dimensional structure of the complexes, and independently by techniques that probe the chemistry of the interactions. However, a severe limitation of such data is that they report only on the endpoints of the binding reaction. Thus, the intermediate events in the binding reaction, such as identification of initial recognition sites and induction of changes in the conformation of the DNA have not been observed or studied. Techniques are under development that utilize visible light to trigger chemical reactions that will enable the time dependence of the reaction of proteins with specific DNA sequences to be examined. This approach will greatly advance our understanding of the molecular mechanism by which proteins recognize and bind to specific sequences of DNA. ***
