MOLECULAR BASIS FOR INHIBITION OF DNA REPAIR INITIATION

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Sites of DNA modification by chemical and physical mutagens are repaired by the excision-repair pathway. This is initiated by DNA glycosylases that cleave the damaged base at its linkage to the sugar. Two such enzymes, uracil-DNA glycosylase from B. subtilis and pyrimidine dimer-DNA glycosylase from T4-infected E. coli are inhibited by the presence of carcinogen-modified non-substrate guanines in DNA. Left unrepaired, uracil in DNA is mutagenic and the pyrimidine dimer both mutagenic and carcinogenic. Therefore carcinogen-modified purines may be mutagenic by inhibition of excision of those glycosylase-sensitive moieties. The basis for this interference is undetermined and the mechanism of recognition of damaged substrates by repair glycosylases is not understood. Therefore the activities of these two purified DNA repair enzymes acting on human alphoid sequences containing more than one form of DNA damage will be studied. The effects of purine adducts of the carcinogens N-acetoxy-N-2-acetylaminofluorene, N-hydroxy-N-2 acetylaminofluorene, dimethyl sulfate and 4-nitroquinoline-1-oxide on recognition and incision of uracil and pyrimidine dimers will be explored. The substrates will be end-labeled and sites of DNA modification analysed by combining chemical and enzymic probes to Maxam-Gilbert sequencing techniques. Sites of enzyme action will be located on these sequencing gels. Sites of enzyme binding to their damaged substrates will be mapped using the photofootprinting technique. This method uses protection of DNA pyrimidines from photochemical damage to demonstrate sites of DNA-protein contacts. The effects of carcinogen-modified purines on the binding of these repair glycosylases will be demonstrated on sequencing gels and quantitated by microdensitometry. The system will be extended to the initiation of excision-repair of DNA damage in cultured human cells. Because carcinogen modification of DNA causes transition from the right-handed B-form to the left-handed Z-form, the enzymic excision of uracil from polymers in these different conformations will be compared. The molecular bases of any differences will be explored by sequencing and photo-footprinting methods. This work will there elucidate the mode of recognition of damaged substrates by DNA glycosylases, demonstrate the effect of damaged purines on such binding, and explore the effects of conformational changes on the repairability of DNA damage.