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 language | English (US) |
|---|---|
| Pages (from-to) | 18790-18799 |
| Number of pages | 10 |
| Journal | Journal of Biological Chemistry |
| Volume | 292 |
| Issue number | 46 |
| DOIs | |
| State | Published - Jan 1 2017 |
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ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Cell Biology
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Mutagenic potential of nitrogen mustard-induced formamidopyrimidine DNA adduct : Contribution of the non-canonical α-anomer. / Minko, Irina; Rizzo, Carmelo J.; Lloyd, Robert (Stephen).
In: Journal of Biological Chemistry, Vol. 292, No. 46, 01.01.2017, p. 18790-18799.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Mutagenic potential of nitrogen mustard-induced formamidopyrimidine DNA adduct
T2 - Contribution of the non-canonical α-anomer
AU - Minko, Irina
AU - Rizzo, Carmelo J.
AU - Lloyd, Robert (Stephen)
PY - 2017/1/1
Y1 - 2017/1/1
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.
UR - http://www.scopus.com/inward/record.url?scp=85034585186&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85034585186&partnerID=8YFLogxK
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 -