The structural details of a DNA molecule, whether subtly or radically different from those of the usual B-form duplex structure, are increasingly recognized as being essential to biological function. For example, the formation of a protein-DNA complex often involves bending or kinking of DNA, and thus the """"""""bendability"""""""" of a particular DNA sequence is likely to be important to its ability to form such a complex. The intermediate in DNA recombination is a four-stranded structure, called the Holliday junction, for which the detailed three- dimensional structure is not known. Knowledge of the role of DNA structure in gene regulation, DNA replication, or recombination will provide new avenues for intervention in disease processes. While X-ray crystallography and Nuclear Magnetic Resonance spectroscopy provide atomic-resolution structural information for DNA, these techniques are limited in their ability to determine the structures of large or complicated DNA molecules in solution. In this proposal new applications of a method for determining DNA structure in solution, involving the chemistry of iron(II), are described. The reaction of the EDTA complex of iron(II) with hydrogen peroxide provides a convenient means for generating the hydroxyl radical (OH) in solution. The hydroxyl radical is perhaps the least-specific agent for cleaving the backbone of DNA. Analysis of the hydroxyl radical cleavage pattern of the DNA of interest provides detailed information on the structure and conformation of the DNA molecule in solution. The following are the Specific Aims of this proposal: (i) to determine which hydrogen(s) of a deoxyribose in the DNA backbone are abstracted in the initial event of DNA cleavage by the hydroxyl radical, by evaluating the kinetic isotope effect on the cleavage reaction using specifically deuterated DNA; (ii) to determine other mechanistic details of the cleavage reaction by product isolation and analysis; (iii) to gain new insight into the sequence-dependence of DNA bending by studying the structural properties of defined-sequence DNA oligonucleotides; (iv) to use a new in vitro selection experiment to isolate new DNA sequences capable of bending; (v) to develop a new method for sequence-specific hydrolysis of DNA, based on a recently-discovered system involving the Flp recombinase enzyme of yeast in combination with hydrogen peroxide.
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