Substrate and Mispairing Properties of 5-formyl-2'-deoxyuridine 5'-triphosphate Assessed by in Vitro DNA Polymerase Reactions

Nucleic Acids Res. 1997 Apr 15;25(8):1570-7. doi: 10.1093/nar/25.8.1570.

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, p K a of the base unit of fdUTP was 8.6, significantly lower than that of parent thymine (p K 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 (p K 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Composition
  • Base Sequence
  • Chromatography, High Pressure Liquid
  • DNA / biosynthesis*
  • DNA / chemistry*
  • DNA Polymerase I / metabolism
  • DNA Replication
  • DNA-Directed DNA Polymerase / metabolism*
  • Deoxyuracil Nucleotides / chemical synthesis
  • Deoxyuracil Nucleotides / isolation & purification
  • Deoxyuracil Nucleotides / metabolism*
  • Escherichia coli / enzymology
  • Hydrogen Bonding
  • Indicators and Reagents
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Molecular Sequence Data
  • Substrate Specificity

Substances

  • 5-formyl-2'-deoxyuridine 5'-triphosphate
  • Deoxyuracil Nucleotides
  • Indicators and Reagents
  • DNA
  • DNA Polymerase I
  • bacteriophage T7 induced DNA polymerase
  • DNA-Directed DNA Polymerase