Mutagenic potential of nitrogen mustard-induced formamidopyrimidine DNA adduct: Contribution of the non-canonical α-anomer

Irina G. Minko, Carmelo J. Rizzo, R. Stephen Lloyd
2017 Journal of Biological Chemistry  
Edited by Patrick Sung Nitrogen mustards (NMs) are DNA-alkylating compounds that represent the earliest anticancer drugs. However, clinical use of NMs is limited because of their own mutagenic properties. The mechanisms of NM-induced mutagenesis remain unclear. The major product of DNA alkylation by NMs is a cationic NM-N7-dG adduct that can yield the imidazole ringfragmented lesion, N 5 -NM-substituted formamidopyrimidine (NM-Fapy-dG). Characterization of this adduct is complicated because it
more » ... dopts different conformations, including both a canonical ␤and an unnatural ␣-anomeric configuration. Although formation of NM-Fapy-dG in cellular DNA has been demonstrated, its potential role in NM-induced mutagenesis is unknown. Here, we created site-specifically modified singlestranded vectors for replication in primate (COS7) or Escherichia coli cells. In COS7 cells, NM-Fapy-dG caused targeted mutations, predominantly G 3 T transversions, with overall frequencies of ϳ11-12%. These frequencies were ϳ2-fold higher than that induced by 8-oxo-dG adduct. Replication in E. coli was essentially error-free. To elucidate the mechanisms of bypass of NM-Fapy-dG, we performed replication assays in vitro with a high-fidelity DNA polymerase, Saccharomyces cerevisiae polymerase (pol) ␦. It was found that pol ␦ could catalyze high-fidelity synthesis past NM-Fapy-dG, but only on a template subpopulation, presumably containing the ␤-anomeric adduct. Consistent with the low mutagenic potential of the ␤-anomer in vitro, the mutation frequency was significantly reduced when conditions for vector preparation were modified to favor this configuration. Collectively, these data implicate the ␣-anomer as a major contributor to NM-Fapy-dG-induced mutagenesis in primate cells. . 2 The abbreviations used are: NM, nitrogen mustard; Fapy, formamidopyrimidine; NM-Fapy-dG, N 5 -NM-substituted formamidopyrimidine dG; Me-Fapy-dG, N 5 -methyl substituted formamidopyrimidine dG; 8-oxo-dG, 8-oxo-7,8-dihydro-2Ј-dG; Endo IV, Endonuclease IV; pol, polymerase.
doi:10.1074/jbc.m117.802520 pmid:28972137 fatcat:y4xbhki4lnbwrfjtj45u3fmorq