Ionizing radiation produces a wide spectrum of damages to the base and sugar moieties of DNA. Enzymes such as endonucleases III and IV, exonuclease III, formamidopyrimidine, uracil and hypoxanthine N- glycosylases from Escherichia coli have been shown to recognize radiation- induced DNA lesions in vitro. The long range goal of this research is to elucidate the enzymatic mechanism(s) by which radiation repair enzymes recognize and remove lesions from damaged DNA. One of the necessary steps in the enzymatic removal of DNA damages is the formation of the protein-DNA lesion complex. The protein-DNA interaction can be elucidated through understanding the mechanism of action of the repair endonucleases as well as elucidating the structural elements involved in the catalysis. The mechanism(s) of action of radiation repair enzymes will be determined by studying the substrate specificity and the reaction kinetics of a number of different Escherichia coli N-glycosylases and endonucleases using oligonucleotides and or circular DNA molecules containing unique base lesions or base lesion analogues as well as modified internucleotide pyrophosphate bonds. The structural elements involved in the formation of protein-DNA complex will be studied by determining the extent of base and phosphate contacts in protein-DNA lesion complexes using DNA footprinting, methylation protection, and ethylation interference techniques. The positional effect of modified internucleotide pyrophosphate linkage on the kinetics of damage recognition will be determined to elucidate the relative contribution of the bases and phosphates in the DNA binding process. The protein structural elements that are essential for binding and/or catalysis will be determined by using amino acid specific chemical modifications, and oligonucleotide directed site specific mutagenesis for the endonucleases. In addition, computer modelling of oligonucleotide containing unique lesions will be performed to increase our understanding of the structure- function relationship the base lesions. Since E. coli AP endonucleases are highly homologous to similar enzymes from yeast and human cells, bacterial repair enzymes provide good model and the reaction mechanisms elucidated should be similar to enzymes purified from higher organisms. The long range goal of this project is to develop specific inhibitors of radiation repair enzymes that could be used in conjunction with radiotherapy, so as to increase the therapeutic efficacy.
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