Misincorporation of dNTPs opposite 1,N2-ethenoguanine and 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine in oligonucleotides by Escherichia coli polymerases I exo- and II exo-, T7 polymerase exo-, human immunodeficiency virus-1 reverse transcriptase, and rat polymerase beta

Biochemistry. 1997 May 20;36(20):6069-79. doi: 10.1021/bi962526v.


1,N2-Ethenoguanine (1,N2-epsilon-Gua) and 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine (HO-ethanoGua) are two modified bases formed in the reaction of DNA with 2-chlorooxirane, the epoxide derivative of vinyl chloride. The oligonucleotides (19-mers), 5'-CAGTGGGTG*TCCGAATTGA-3', were prepared, with each of these modified bases substituted for G at G*. HO-ethanodeoxyguanosine exists predominantly as a mixture of diastereomers of the closed cyclic hemiaminal form, 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine, shown by H2(18)O experiments to be in equilibrium with the open form, N2-(2-oxoethyl)Gua. Both adducts retarded the 3'-extension of a complementary 10-mer primer by all of the polymerases examined, but in every case, some full-length product was obtained. Nucleotide sequence analysis indicated misincorporation of dGTP and dATP across from both 1,N2-epsilon-Gua and HO-ethanoGua, with the extent varying considerably among the polymerases. Similar results were obtained when the abilities of the polymerases to incorporate a single dNTP were evaluated. In addition, -1 and -2 base frame shifts were detected with both 1,N2-epsilon-Gua and HO-ethanoGua with some of the polymerases. Steady-state kinetic experiments with Escherichia coli polymerase I exo- and T7 polymerase exo-/thioredoxin showed large decreases in k(cat) for all dNTP incorporations compared to the normal G x dCTP pair and high misincorporation frequencies for dATP and dGTP with both adducts (compared to dCTP). Collectively, the results indicate that both of these adducts have considerable miscoding potential with some of these polymerases, that there are a number of differences between the 1,N2-epsilon-Gua and HO-ethanoGua adducts (which formally differ only in the presence of the elements of water), and that misincorporation of dNTPs at a single modified base can vary considerably among different polymerases even in the absence of exonuclease activity.

Publication types

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

MeSH terms

  • Carcinogens / metabolism
  • DNA-Directed DNA Polymerase / metabolism*
  • Deoxyadenine Nucleotides / metabolism
  • Deoxyguanine Nucleotides / metabolism
  • Deoxyribonucleotides / chemistry
  • Deoxyribonucleotides / metabolism*
  • Guanine / analogs & derivatives*
  • Guanine / chemistry
  • Guanine / metabolism
  • Imidazoles / chemistry
  • Imidazoles / metabolism*
  • Mutagenesis
  • Oligodeoxyribonucleotides / biosynthesis
  • Purines / chemistry
  • Purines / metabolism*
  • Transcription, Genetic*


  • Carcinogens
  • Deoxyadenine Nucleotides
  • Deoxyguanine Nucleotides
  • Deoxyribonucleotides
  • Imidazoles
  • Oligodeoxyribonucleotides
  • Purines
  • 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo(1,2-a)purine
  • 1,(N2)-ethenoguanine
  • Guanine
  • deoxyguanosine triphosphate
  • DNA-Directed DNA Polymerase