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It was shown in 2000 that treatment of deoxyguanosine (dG) with the carcinogen diisopropyltriazene results in N2-isopropyl-dG (N2IdG) adducts. Little is known about the biological consequences of N2IdG in human DNA. To study this issue, duplex oligonucleotides d(pTCGAGACTTCXAAGGGTT), X= chemically synthesized N2IdG, annealed to d(pCCGGAACCCTTC GAAGTC) or d(pCCGGAACCCUUCGAAGUC) were ligated into the supF tRNA gene of the human cell mutation reporting plasmid pLS1d. These constructs containing single N2IdG were then either transformed directly into E. coli strain MBM7070, which distinguishes wild type from mutant supF by the presence of beta galactosidase activity, or transfected into human embryonic kidney 293 cells. Forty eight hours after 293 cell transfection, allowing time for replication and repair, plasmid DNA was recovered and subsequently transformed into MBM7070 cells. Mutant fractions and the types of mutations were analyzed. No significant difference in the mutant fraction of N2IdG vs. dG-containing constructs without deoxyuridine (dU) in the complementary strand was observed after replication in either 293 cells or E. coli strain MBM7070. However, when dU was present in the complementary strand, the mutant fraction for N2IdG-containing construct was 4.7% vs. 0.3% for dG-containing construct after replication in E. coli strain MBM7070, a 15-fold N2IdG -mediated enhancement (p = .0005). Similarly, when dU-containing plasmids were analyzed the mutant fraction for N2IdG-containing construct was 2.2% vs. 0.2% for dG-containing construct after replication in human 293 cells, an 11-fold N2IdG -mediated enhancement. Twenty four independent mutants have been sequenced from E. coli; the mutation spectrum is approximately equally distributed between G:C → T:A transitions (7/24, 29.2%) at N2IdG, single base deletions (6/24, 25.0%), and G:C → A:T transversions (4/24,16.7%). These data indicate that N2IdG is mutagenic when serving as a template base in human DNA, producing transitions, point deletions, and transversions.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]