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 - Funding Information:
This work was supported by National Institutes of Health, NCI Grants T32 CA106195, P01 CA160032, and P30 CA068485 and NIEHS Grants P30 ES000267 and T32 ES0007060. The authors declare that they have no con-flicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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
VL - 292
SP - 18790
EP - 18799
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 46
ER -