Benzo[a]pyrene is a potent mutagen/carcinogen found ubiquitously in the environment. It is metabolically activated inside cells to its (+)-anti-7,8-diol-9,10-epoxide giving adducts principally at N2-Gua ([+ta]-B[a]P-N2-dG). Applicant showed that (+)-anti-B[a]PDE induces base substitution, frameshifts, insertions and deletions (studied in E. coli). The question he is addressing is: how does (+)-anti-B[a]PDE induce such a diverse array of mutations? His working hypothesis is that adduct mutational complexity is due to adduct conformational complexity and that the major adduct ([+ta]-B[a]P-N2-Gua) is able to induce the majority of these mutations. Furthermore, adduct conformation may be controlled by various factors, notably, DNA sequence context. We have generated additional data to support his working hypothesis, and have found that [+ta]-B[a]P-N2-dG principally follows the G->T mutational pathway in 5'-TGC sequences (>95 percent), but principally the G->A mutational pathway in a 5'-AGA ( about95 percent) and a 5'-CGY ( about80 percent) sequence context. A correlation between his mutagenesis findings and molecular modeling studies (funded separately) suggested a conformational hypothesis for the induction of G->T vs. G->A mutation. Preliminary findings suggest that this hypothesis is probably not correct. He believes that more data are needed on the effect of DNA sequence context on [+ta]-B[a]P-N2-dG mutagenesis before another hypothesis can be proposed. In addition, he believes that one must understand what polymerase is involved in mutagenic bypass. To this end, three specific aims are proposed. (1) The mutagenic patterns for [+ta]-B[a]P-N2-dG in a 5'-TGT vs. a 5'-UGT context and a 5'-m5CGT- vs. a 5'-CGT context will be compared to evaluate the role of a methyl group on the base to the 5'-side of the adduct to influence the pattern of mutagenesis.
This aim relates to a hypothesis for why G->T mutations are high in 5'-TG sequences. (2) [+ta]-B[a]P-N2-dG will be studied in all combinations of sequence contexts 5'-XGY from which sequence context rules for mutagenesis will emerge. These rules (in combination with structural/conformational studies) will be used to generate new ideas for how adduct conformations might induce (e.g.) G->T vs. G->A mutations. The vector in this study will permit the applicant to do studies in both E. coli and human cells in culture. (3) Investigate what E. coli DNA polymerase(s) (notably, DNA polymerases II, IV, V, or UVM) is (are) responsible for G->T vs. G->A mutations for [+ta]-B[a]P-N2-dG.
Showing the most recent 10 out of 41 publications
