FOOTPRINTING WITH IRON(II)-GENERATED HYDROXYL RADICAL

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The main objective of the proposed experiments is to provide detailed structural information on protein-DNA complexes in solution. The experimental method to be used make use of chemistry of iron (II) with hydrogen peroxide to generate the hydroxyl radical. This very small and reactive molecule reacts with the backbone of DNA, leaving gaps in the DNA strand at the site of reaction. Bound protein protects the DNA from cleavage at the points of protein-DNA contact. This observation forms the basis of the newly-developed hydroxy radical "footprinting" method that will be used in the proposed experiments. There are three specific goals of the research proposed: 1) to use X-ray crystal structures of DNA- protein complexes to "calibrate" the hydroxyl radical footprinting method, in order to determine the structural features of protein-DNA complexes to which the hydroxyl radical cleavage reaction is sensitive; 2) to use the hydroxyl radical footprinting method to determine structural changes that occur during initiation of DNA replication in SV40; and 3) to study "higher-order" protein-DNA complexes that are made up of multiple proteins bound to DNA. The three higher-order systems to be studied are the bacteriophage lambda repressor-operator system, the transcription complex for the 5S genes of Xenopus that includes TFIIIA, TFIIIB and TFIIIC, and the simultaneous interaction of the UBX protein of Drosophila with two binding sites near its own gene. The ability of hydroxyl radical footprinting to determine structural details for complicated protein-DNA systems, as exemplified in goal 3), provides a new way to study the components of the genetic systems of the living cell.