FOOTPRINTING WITH IRON (II)-GENERATED HYDROXYL RADICAL

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The reaction of iron(II) EDTA with hydrogen peroxide produces the hydroxyl radical, which has proven to be a powerful and widely- used chemical probe of the structure of protein-DNA and protein- RNA complexes. The hydroxyl radical also is produced by the interaction of ionizing radiation with water, and as such is the proximate chemical species that mediates radiation damage to DNA. The long-term goal of this project is to make use of the chemistry of the hydroxyl radical, produced either using the iron(II) EDTA/hydrogen peroxide system or by gamma irradiation, to make high-resolution chemical "images" of complicated functioning protein-DNA complexes. Systems to be studied include actively-transcribing RNA polymerase, a set of copper metalloregulatory factors from yeast, and the Z-DNA-binding, RNA -editing enzyme ADAR1. A collateral goal of the project is to use new knowledge on the structure and properties of DNA damaged by the hydroxyl radical to isolate and characterize cellular proteins that initially recognize radiation damage to the DNA backbone. The Specific Aims of the project are: (1) to use the hydroxyl radical as a chemical probe to dissect the interactions that the DNA-binding subdomains of the yeast copper metalloregulatory factors Amt1, Ace1, and Mac1 make with DNA; (2) to prepare a set of three specifically-lesioned DNA oligonucleotides and use them to isolate eukaryotic proteins which recognize oxidative damage to the DNA backbone that is induced by ionizing radiation; (3) to use the results of missing nucleoside experiments on transcribing RNA polymerase performed in the past grant period to prepare specifically-gapped template DNA molecules for analysis of the effect of template structure on transcription. As well, DNA templates with bent and straight adenine tracts will be prepared for studies on the effect of global DNA structure on transcription; (4) to perform hydroxyl radical footprinting experiments on the complex of the RNA editing enzyme ADAR1 with left-handed Z-DNA, with the eventual goal of performing chemical probe experiments to define the structure of a functioning RNA editing system. The results of this project will provide new information on how cells recognize damage to the genome cause by ionizing radiation. As well, the studies to be undertaken on RNA polymerase, the yeast copper metalloregulatory factors, and ADAR1 will give new structural insight into some of the complicated protein-DNA "machines" which the cell uses to decode the information in the genome.