Oxidation of Thymine to 5-Formyluracil in DNA Promotes Misincorporation of dGMP and Subsequent Elongation of a Mismatched Primer Terminus by DNA Polymerase

Aya Masaoka, Hiroaki Terato, Mutsumi Kobayashi, Yoshihiko Ohyama, Hiroshi Ide

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

5-Formyluracil (fU) is a major oxidative thymine lesion generated by ionizing radiation and reactive oxygen species. In the present study, we have assessed the influence of fU on DNA replication to elucidate its genotoxic potential. Oligonucleotide templates containing fU at defined sites were replicated in vitro by Escherichia coli DNA polymerase I Klenow fragment deficient in 3′-5′-exonuclease. Gel electrophoretic analysis of the reaction products showed that fU constituted very weak replication blocks to DNA synthesis, suggesting a weak to negligible cytotoxic effect of this lesion. However, primer extension assays with a single dNTP revealed that fU directed incorporation of not only correct dAMP but also incorrect dGMP, although much less efficiently. No incorporation of dCMP and dTMP was observed. When fU was substituted for T in templates, the incorporation efficiency of dAMP (f A = Vmax/Km) decreased to 1/4 to 1/2, depending on the nearest neighbor base pair, and that of dGMP (fG) increased 1.1-5.6-fold. Thus, the increase in the replication error frequency (fG/fA for fU versus T) was 3.1-14.3-fold. The misincorporation rate of dGMP opposite fU (pKa = 8.6) but not T (pKa = 10.0) increased with pH (7.2-8.6) of the reaction mixture, indicating the participation of the ionized (or enolate) form of fU in the mispairing with G. The resulting mismatched fU:G primer terminus was more efficiently extended than the T:G terminus (8.2-11.3-fold). These results show that when T is oxidized to fU in DNA, fU promotes both misincorporation of dGMP at this site and subsequent elongation of the mismatched primer, hence potentially mutagenic.

Original languageEnglish
Pages (from-to)16501-16510
Number of pages10
JournalJournal of Biological Chemistry
Volume276
Issue number19
DOIs
Publication statusPublished - May 11 2001
Externally publishedYes

Fingerprint

Thymine
DNA-Directed DNA Polymerase
Elongation
Oxidation
DNA
DNA Polymerase I
2'-deoxyguanosine 5'-phosphate
5-formyluracil
Exonucleases
Ionizing radiation
Ionizing Radiation
DNA Replication
Reaction products
Oligonucleotides
Base Pairing
Escherichia coli
Assays
Reactive Oxygen Species
Gels

ASJC Scopus subject areas

  • Biochemistry

Cite this

Oxidation of Thymine to 5-Formyluracil in DNA Promotes Misincorporation of dGMP and Subsequent Elongation of a Mismatched Primer Terminus by DNA Polymerase. / Masaoka, Aya; Terato, Hiroaki; Kobayashi, Mutsumi; Ohyama, Yoshihiko; Ide, Hiroshi.

In: Journal of Biological Chemistry, Vol. 276, No. 19, 11.05.2001, p. 16501-16510.

Research output: Contribution to journalArticle

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abstract = "5-Formyluracil (fU) is a major oxidative thymine lesion generated by ionizing radiation and reactive oxygen species. In the present study, we have assessed the influence of fU on DNA replication to elucidate its genotoxic potential. Oligonucleotide templates containing fU at defined sites were replicated in vitro by Escherichia coli DNA polymerase I Klenow fragment deficient in 3′-5′-exonuclease. Gel electrophoretic analysis of the reaction products showed that fU constituted very weak replication blocks to DNA synthesis, suggesting a weak to negligible cytotoxic effect of this lesion. However, primer extension assays with a single dNTP revealed that fU directed incorporation of not only correct dAMP but also incorrect dGMP, although much less efficiently. No incorporation of dCMP and dTMP was observed. When fU was substituted for T in templates, the incorporation efficiency of dAMP (f A = Vmax/Km) decreased to 1/4 to 1/2, depending on the nearest neighbor base pair, and that of dGMP (fG) increased 1.1-5.6-fold. Thus, the increase in the replication error frequency (fG/fA for fU versus T) was 3.1-14.3-fold. The misincorporation rate of dGMP opposite fU (pKa = 8.6) but not T (pKa = 10.0) increased with pH (7.2-8.6) of the reaction mixture, indicating the participation of the ionized (or enolate) form of fU in the mispairing with G. The resulting mismatched fU:G primer terminus was more efficiently extended than the T:G terminus (8.2-11.3-fold). These results show that when T is oxidized to fU in DNA, fU promotes both misincorporation of dGMP at this site and subsequent elongation of the mismatched primer, hence potentially mutagenic.",
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T1 - Oxidation of Thymine to 5-Formyluracil in DNA Promotes Misincorporation of dGMP and Subsequent Elongation of a Mismatched Primer Terminus by DNA Polymerase

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AU - Terato, Hiroaki

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AU - Ide, Hiroshi

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N2 - 5-Formyluracil (fU) is a major oxidative thymine lesion generated by ionizing radiation and reactive oxygen species. In the present study, we have assessed the influence of fU on DNA replication to elucidate its genotoxic potential. Oligonucleotide templates containing fU at defined sites were replicated in vitro by Escherichia coli DNA polymerase I Klenow fragment deficient in 3′-5′-exonuclease. Gel electrophoretic analysis of the reaction products showed that fU constituted very weak replication blocks to DNA synthesis, suggesting a weak to negligible cytotoxic effect of this lesion. However, primer extension assays with a single dNTP revealed that fU directed incorporation of not only correct dAMP but also incorrect dGMP, although much less efficiently. No incorporation of dCMP and dTMP was observed. When fU was substituted for T in templates, the incorporation efficiency of dAMP (f A = Vmax/Km) decreased to 1/4 to 1/2, depending on the nearest neighbor base pair, and that of dGMP (fG) increased 1.1-5.6-fold. Thus, the increase in the replication error frequency (fG/fA for fU versus T) was 3.1-14.3-fold. The misincorporation rate of dGMP opposite fU (pKa = 8.6) but not T (pKa = 10.0) increased with pH (7.2-8.6) of the reaction mixture, indicating the participation of the ionized (or enolate) form of fU in the mispairing with G. The resulting mismatched fU:G primer terminus was more efficiently extended than the T:G terminus (8.2-11.3-fold). These results show that when T is oxidized to fU in DNA, fU promotes both misincorporation of dGMP at this site and subsequent elongation of the mismatched primer, hence potentially mutagenic.

AB - 5-Formyluracil (fU) is a major oxidative thymine lesion generated by ionizing radiation and reactive oxygen species. In the present study, we have assessed the influence of fU on DNA replication to elucidate its genotoxic potential. Oligonucleotide templates containing fU at defined sites were replicated in vitro by Escherichia coli DNA polymerase I Klenow fragment deficient in 3′-5′-exonuclease. Gel electrophoretic analysis of the reaction products showed that fU constituted very weak replication blocks to DNA synthesis, suggesting a weak to negligible cytotoxic effect of this lesion. However, primer extension assays with a single dNTP revealed that fU directed incorporation of not only correct dAMP but also incorrect dGMP, although much less efficiently. No incorporation of dCMP and dTMP was observed. When fU was substituted for T in templates, the incorporation efficiency of dAMP (f A = Vmax/Km) decreased to 1/4 to 1/2, depending on the nearest neighbor base pair, and that of dGMP (fG) increased 1.1-5.6-fold. Thus, the increase in the replication error frequency (fG/fA for fU versus T) was 3.1-14.3-fold. The misincorporation rate of dGMP opposite fU (pKa = 8.6) but not T (pKa = 10.0) increased with pH (7.2-8.6) of the reaction mixture, indicating the participation of the ionized (or enolate) form of fU in the mispairing with G. The resulting mismatched fU:G primer terminus was more efficiently extended than the T:G terminus (8.2-11.3-fold). These results show that when T is oxidized to fU in DNA, fU promotes both misincorporation of dGMP at this site and subsequent elongation of the mismatched primer, hence potentially mutagenic.

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