C8-Guanine Adduct-Induced Stabilization of a −1 Frame Shift Intermediate in a Nonrepetitive DNA Sequence

Abstract

The mechanism of frame shift mutagenesis induced by N-(deoxyguanosin-8-yl)-1-aminopyrene, the major DNA adduct formed by the carcinogen 1-nitropyrene, was investigated by thermal melting studies of a 13-mer in which the adduct was flanked by a 5' and a 3' C. Compared to the unmodified 13-mer, the adduct destabilized the duplex by 4-5 kcal/mol, and the DeltaDeltaG value remained approximately the same regardless of which base was placed opposite the adduct. In contrast, deletion of the base opposite the adduct stabilized the duplex by nearly 4 kcal/mol. The adduct in the same sequence context was inserted into a bacteriophage M13 DNA containing the simian virus 40 origin of replication. The constructed DNA template was replicated in vitro with extracts from normal human fibroblasts. The adduct was not removed from the progeny DNA following bidirectional semiconservative replication, which suggests that it had been bypassed, rather than repaired, by the cell extract. When newly replicated bacteriophage was evaluated for mutations in the region of the modified G, most contained a G at the adduct site, indicating error-free replication. A small number of mutants ( approximately 2 x 10(-3)) were detected, all of which contained a targeted G.C base pair deletion. This suggests a relationship between the thermodynamic stability of the adduct in DNA and the errors that occurred during replicative bypass by the human DNA polymerases.