Replication of the base pair 6-thioguanine/5-methyl-2-pyrimidine with the large Klenow fragment of Escherichia coli DNA polymerase I

Biochemistry. 1993 Mar 30;32(12):3047-57. doi: 10.1021/bi00063a016.


The kinetics and the fidelity of replication of the base pair 6-thioguanine (Gs)/5-methyl-2-pyrimidinone (Th) have been determined by using defined oligomers with the large Klenow fragment of Escherichia coli DNA polymerase I. The insertion efficiency, Vmax/Km (min-1 microM-1), of Th opposite Gs is 1.5 and the insertion efficiency of Gs opposite Th is 0.7. By comparison, the insertion efficiencies of C opposite G and G opposite C are 0.5 and 1.5. The insertion efficiency of the next base, A opposite T, is 2 times greater after the base pair Gs/Th than after G/C. The fidelity of replication with respect to thymine and adenine has misinsertion frequencies, or ratios of the insertion efficiency of the "wrong" base to the "right" base, of 7 x 10(-4) for T opposite Gs (T/Gs), 4 x 10(-6) for T/Th, and a maximum stable misinsertion frequency of 4 x 10(-4) for A/Th. No detectable elongation occurs after an A is inserted opposite a Gs. These values are similar to the misinsertion frequencies of G and C with T and A. The maximum stable misinsertion frequencies with G and C are 4 x 10(-2) for G/Th, 3 x 10(-2) -7 x 10(-3) for Gs/C, and 2.6 x 10(-1) for C/Gs, and the misinsertion frequency is < 1 x 10(-3) for Th/G. The kinetics results and molecular modeling suggest modifications to the Gs/Th base pair that may provide higher levels of fidelity of replication with respect to C and G.

MeSH terms

  • Base Composition
  • DNA Polymerase I / metabolism*
  • DNA Replication
  • Deoxyadenine Nucleotides / metabolism
  • Deoxyguanine Nucleotides / metabolism
  • Escherichia coli / enzymology*
  • Exodeoxyribonuclease V
  • Exodeoxyribonucleases / metabolism
  • Kinetics
  • Mathematics
  • Models, Biological
  • Pyrimidinones / metabolism*
  • Templates, Genetic
  • Thioguanine / metabolism*
  • Thymine Nucleotides / metabolism


  • Deoxyadenine Nucleotides
  • Deoxyguanine Nucleotides
  • Pyrimidinones
  • Thymine Nucleotides
  • 5-methyl-2-pyrimidinone
  • deoxyguanosine triphosphate
  • DNA Polymerase I
  • Exodeoxyribonucleases
  • Exodeoxyribonuclease V
  • Thioguanine
  • 2'-deoxyadenosine triphosphate
  • thymidine 5'-triphosphate