Endonuclease III (endo III) and exonuclease III (exo III) of Escherichia coli represent a class of radiation raprir endonucleases/glycosylases that recognize DNA substrates damaged by ionizing radiation. The goal of this research will be to investigate the mechanism of action of these two enzymes. The first approach will be to chemically modify existing apurinic sites or urea residues directly on DNA molecule to form novel substrates that are analogs of radiation-induced base damages. In contrast to the current method of producing radiation-induced or oxidative DNA base modifications in which a spectrum of damages are produced, this approach will enable the production of unique DNA modifications. Chemically modified apurinic sites or urea residues with known chemical structures will be tested for their ability to serve as substrates for endo III and exo III. A comparative kinetic study will then be performed with these substrates to deduce the possible mechanism of action of these two radiation repair enzymes. The second approach will involve the direct analysis of the products of the endo III and exo III reactions. The analysis of acid soluble products as well as residues left on the DNA will enable us to deduce the possible chemistry of cleavage of the phospodiester bond adjacent to the damage. The third approach will involve the isolation of stable enzyme-damage (DNA) complexes, and the direct amino acid sequencing of the active sites of these enzymes. These studies with endo III and exo III will be extended to other radiation repair endonucleases both from yeast and E. coli, so that the general mode of action of these endonucleases/glycosylases can be understood. DNA damages produced by free radical reactions can lead to mutation, cancer and aging thus the study of their repair is important for the understanding of these fundamental biological processes.
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