FOOTPRINTING WITH IRON(II)-GENERATED HYDROXYL RADICAL

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For its biological activity to be expressed, DNA must be associated with protein. Deeper understanding of the interactions of proteins with DNA will provide new avenues for disease intervention. The long term goal of this work is to gain new information on the complicated protein-DNA complexes that exist in the cell. New methods have been developed to study the structure and energetics of DNA-protein complexes. These methods depend on the chemistry of iron(H) EDTA with hydrogen peroxide as a convenient means of generating the hydroxyl radical in solution. The hydroxyl radical cleaves the DNA backbone by abstracting a hydrogen atom from a deoxyribose, leaving a single-stranded gap in the DNA. This chemistry can be used to make very high resolution "footprints" of proteins bound to DNA. Another method based on hydroxyl radical chemistry is the recently-developed Missing Nucleoside Experiment, which provides direct information on the energetically important contacted that a protein makes with its DNA binding site. The Specific Aims of this application are: (1) to develop the Missing Nucleoside Experiment into a quantitative measure of the free energy of interaction of each nucleotide in a DNA binding site with its cognate protein. (2) to use hydroxyl radical footprinting and the Missing Nucleoside Experiment to characterize the DNA binding of a series of mutated homeodomain proteins, which are involved in specifying developmental pathways in higher organisms. (3) to use chemical probe experiments, including the Missing Nucleoside Experiment, to determine the structural features of RNA polymerase-DNA complexes engaged in transcription initiation and elongation. (4) to develop a new method for making high resolution footprints of DNA-protein complexes in living cells, using gamma radiation to produce hydroxyl radical in vivo. This new in vivo footprinting methods will be applied to characterizing the tissue- specific expression of the growth hormone gene.