TY - JOUR
T1 - Mutagenic potential of nitrogen mustard-induced formamidopyrimidine DNA adduct
T2 - Contribution of the non-canonical α-anomer
AU - Minko, Irina G.
AU - Rizzo, Carmelo J.
AU - Stephen Lloyd, R.
N1 - Publisher Copyright:
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2017/11/17
Y1 - 2017/11/17
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.M117.802520
DO - 10.1074/jbc.M117.802520
M3 - Article
C2 - 28972137
AN - SCOPUS:85034585186
SN - 0021-9258
VL - 292
SP - 18790
EP - 18799
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 46
ER -