PYRIDYLOXOBUTYLATION OF GUANINE RESIDUES BY CETOXYMETHYL)NITROSAMINO]-1-(3-PYRIDYL)-1-BUTANONE GENERATES SUBSTRATES OF O-6-ALKYLGUANINE-DNAALKYLTRANSFERASE

Pyridyloxobutylation of DNA yields adducts that react with O-6-alkylgu anine-DNA alkyltransferase (AGT) to prevent the repair of O-6-methylgu anine (O-6-mG). The chemical characterization of pyridyloxobutyl adduc ts has been confounded by their instability under DNA hydrolysis condi tions. They decompose to 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) duri ng the chemical or enzymatic hydrolysis of DNA. The goal of these stud ies was to determine which bases are pyridyloxobutylated to form AGT-r eactive adducts. The model pyridyloxobutylating agent, cetoxymethyl)ni trosamino]-1-(3-pyridyl)-1-butanone (NNKOAc), was reacted with either poly(dAdT) or poly(dGdC) to generate DNA substrates for reaction with AGT. Only the pyridyloxobutylated poly(dGdC) was able to prevent the a bility of partially purified rat liver AGT to repair O-6-mG. These res ults paralleled those obtained for the corresponding methylated substr ates. These studies are consistent with the pyridyloxobutylation of GC base pairs and not AT base pairs in the DNA to generate a substrate f or AGT. In order to distinguish between the formation of reactive addu cts at C residues versus G residues, two oligomers were designed that were complementary to one another. One oligomer contained A, T, and G residues, whereas its complement contained T, A, and C residues. Only the dG-containing oligomer reacted with NNKOAc to generate an AGT-reac tive adduct, again paralleling the results obtained for a methylating agent; These results demonstrate that pyridyloxobutylation of only gua nine residues produces adducts that react with AGT. These AGT-reactive guanine adducts are relatively stable within DNA, with a half-life of 1-2 weeks at 37 degrees C. They represent up to 70% of the total HPB- releasing adducts in the NNKOAc-treated DNA. We postulate that a poten tial AGT-reactive adduct is an O-6-(pyridyloxobutyl)guanine adduct.