Substrate and mispairing properties of 5-formyl-2'-deoxyuridine 5'-triphosphate assessed by in vitro DNA polymerase reactions

Mitsuo Yoshida, Keisuke Makino, Hajime Morita, Hiroaki Terato, Yoshihiko Ohyama, Hiroshi Ide

Research output: Contribution to journalArticle

60 Citations (Scopus)

Abstract

5-Formyluracil (fU) is one of the thymine lesions produced by reactive oxygen radicals in DNA and its constituents. In this work, 5-formyl-2'-deoxyuridine 5'-triphosphate (fdUTP) was chemically synthesized and extensively purified by HPLC. The electron withdrawing 5-formyl group facilitated ionization of fU. Thus, pK(a) of the base unit of fdUTP was 8.6, significantly lower than that of parent thymine (pK(a) = 10.0 as dTMP). fdUTP efficiently replaced dTTP during DNA replication catalyzed by Escherichia coli DNA polymerase I (Klenow fragment), T7 DNA polymerase (3'-5' exonuclease free) and Taq DNA polymerase. fU-specific cleavage of the replication products by piperidine revealed that when incorporated as T, incorporation of fU was virtually uniform, suggesting minor sequence context effects on the incorporation frequency of fdUTP. fdUTP also replaced dCTP, but with much lower efficiency than that for dTTP. The substitution efficiency for dCTP increased with increasing pH from 7.2 to 9.0. The parallel correlation between ionization of the base unit of fdUTP (pK(a) = 8.6) and the substitution efficiency for dCTP strongly suggests that the base-ionized form of fdUTP is involved in mispairing with template G. These data indicate that fU can be specifically introduced into DNA as unique lesions by in vitro DNA polymerase reactions. In addition, fU is potentially mutagenic since this lesion is much more prone to form mispairing with G than parent thymine.

Original languageEnglish
Pages (from-to)1570-1577
Number of pages8
JournalNucleic Acids Research
Volume25
Issue number8
DOIs
Publication statusPublished - Apr 15 1997
Externally publishedYes

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DNA-Directed DNA Polymerase
Thymine
DNA Polymerase I
Taq Polymerase
Exonucleases
DNA
DNA Replication
deoxyuridine triphosphate
5-formyluracil
In Vitro Techniques
5-formyl-2'-deoxyuridine 5'-triphosphate
Reactive Oxygen Species
High Pressure Liquid Chromatography
Electrons
Escherichia coli
2'-deoxycytidine 5'-triphosphate

ASJC Scopus subject areas

  • Genetics

Cite this

Substrate and mispairing properties of 5-formyl-2'-deoxyuridine 5'-triphosphate assessed by in vitro DNA polymerase reactions. / Yoshida, Mitsuo; Makino, Keisuke; Morita, Hajime; Terato, Hiroaki; Ohyama, Yoshihiko; Ide, Hiroshi.

In: Nucleic Acids Research, Vol. 25, No. 8, 15.04.1997, p. 1570-1577.

Research output: Contribution to journalArticle

Yoshida, Mitsuo ; Makino, Keisuke ; Morita, Hajime ; Terato, Hiroaki ; Ohyama, Yoshihiko ; Ide, Hiroshi. / Substrate and mispairing properties of 5-formyl-2'-deoxyuridine 5'-triphosphate assessed by in vitro DNA polymerase reactions. In: Nucleic Acids Research. 1997 ; Vol. 25, No. 8. pp. 1570-1577.
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N2 - 5-Formyluracil (fU) is one of the thymine lesions produced by reactive oxygen radicals in DNA and its constituents. In this work, 5-formyl-2'-deoxyuridine 5'-triphosphate (fdUTP) was chemically synthesized and extensively purified by HPLC. The electron withdrawing 5-formyl group facilitated ionization of fU. Thus, pK(a) of the base unit of fdUTP was 8.6, significantly lower than that of parent thymine (pK(a) = 10.0 as dTMP). fdUTP efficiently replaced dTTP during DNA replication catalyzed by Escherichia coli DNA polymerase I (Klenow fragment), T7 DNA polymerase (3'-5' exonuclease free) and Taq DNA polymerase. fU-specific cleavage of the replication products by piperidine revealed that when incorporated as T, incorporation of fU was virtually uniform, suggesting minor sequence context effects on the incorporation frequency of fdUTP. fdUTP also replaced dCTP, but with much lower efficiency than that for dTTP. The substitution efficiency for dCTP increased with increasing pH from 7.2 to 9.0. The parallel correlation between ionization of the base unit of fdUTP (pK(a) = 8.6) and the substitution efficiency for dCTP strongly suggests that the base-ionized form of fdUTP is involved in mispairing with template G. These data indicate that fU can be specifically introduced into DNA as unique lesions by in vitro DNA polymerase reactions. In addition, fU is potentially mutagenic since this lesion is much more prone to form mispairing with G than parent thymine.

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