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

Irina Minko, Carmelo J. Rizzo, Robert (Stephen) Lloyd

Research output: Contribution to journalArticle

6 Scopus citations

Abstract

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, N5-NM-substituted formamidopyrimidine (NM-Fapy-dG). Characterization of this adduct is complicated because it adopts different conformations, including both a canonical β- and an unnatural α-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 → T transversions, with overall frequencies of 11-12%. These frequencies were ~2-fold higher than that induced by 8-oxo-dG adduct. Replication in E. coli was essentially error-free. To elucidate themechanismsof bypass of NM-Fapy-dG, we performed replication assays in vitro with a high-fidelity DNA polymerase, Saccharomyces cerevisiae polymerase (pol) δ. It was found that pol δ could catalyze high-fidelity synthesis past NMFapy-dG, but only on a template subpopulation, presumably containing the β-anomeric adduct. Consistent with the low mutagenic potential of the β-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 α-anomeras amajorcontributor toNM-Fapy-dG-induced mutagenesis in primate cells.

Original languageEnglish (US)
Pages (from-to)18790-18799
Number of pages10
JournalJournal of Biological Chemistry
Volume292
Issue number46
DOIs
StatePublished - Jan 1 2017

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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