Miscoding potential of the N2-ethyl-2'-deoxyguanosine DNA adduct by the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I

Biochemistry. 2001 Apr 3;40(13):4106-14. doi: 10.1021/bi002719p.


Acetaldehyde, a major metabolite of ethanol, reacts with dG residues in DNA, resulting in the formation of the N(2)-ethyl-2'-deoxyguanosine (N(2)-Et-dG) adduct. This adduct has been detected in lymphocyte DNA of alcohol abusers. To explore the miscoding property of the N(2)-Et-dG DNA adduct, phosphoramidite chemical synthesis was used to prepare site-specifically modified oligodeoxynucleotides containing a single N(2)-Et-dG. These N(2)-Et-dG-modified oligodeoxynucleotides were used as templates for primer extension reactions catalyzed by the 3' --> 5' exonuclease-free (exo(-)) Klenow fragment of Escherichia coli DNA polymerase I. The primer extension was retarded one base prior to the N(2)-Et-dG lesion and opposite the lesion; however, when the enzyme was incubated for a longer time or with increased amounts of this enzyme, full extension occurred. Quantitative analysis of the fully extended products showed the preferential incorporation of dGMP and dCMP opposite the N(2)-Et-dG lesion, accompanied by a small amounts of dAMP and dTMP incorporation and one- and two-base deletions. Steady-state kinetic studies were also performed to determine the frequency of nucleotide insertion opposite the N(2)-Et-dG lesion and chain extension from the 3' terminus from the dN.N(2)-Et-dG (N is C, A, G, or T) pairs. These results indicate that the N(2)-Et-dG DNA adduct may generate G --> C transversions in living cells. Such a mutational spectrum has not been detected with other methylated dG adducts, including 8-methyl-2'-deoxyguanosine, O(6)-methyl-2'-deoxyguanosine, and N(2)-methyl-2'-deoxyguanosine. In addition, N(2)-ethyl-2'-deoxyguanosine triphosphate (N(2)-Et-dGTP) was efficiently incorporated opposite a template dC during DNA synthesis catalyzed by the exo(-) Klenow fragment. The utilization of N(2)-Et-dGTP was also determined by steady-state kinetic studies. N(2)-Et-dG DNA adducts are also formed by the incorporation of N(2)-Et-dGTP into DNA and may cause mutations, leading to the development of alcohol- and acetaldehyde-induced human cancers.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • DNA Adducts / genetics*
  • DNA Adducts / metabolism
  • DNA Polymerase I / genetics*
  • DNA Polymerase I / metabolism
  • DNA Primers / metabolism
  • DNA, Bacterial / metabolism
  • Deoxyguanine Nucleotides / genetics*
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Exodeoxyribonucleases / metabolism
  • Genetic Code
  • Intercalating Agents / metabolism
  • Kinetics
  • Oligodeoxyribonucleotides / chemical synthesis
  • Oligodeoxyribonucleotides / metabolism
  • Templates, Genetic


  • DNA Adducts
  • DNA Primers
  • DNA, Bacterial
  • Deoxyguanine Nucleotides
  • Intercalating Agents
  • N(2)-ethyl-2'-deoxyguanosine triphosphate
  • Oligodeoxyribonucleotides
  • DNA Polymerase I
  • Exodeoxyribonucleases