Replication of the base pair 6-thioguanine/5-methyl-2-pyrimidine with the large Klenow fragment of Escherichia coli DNA polymerase I.
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.
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Biology Department, Temple University, Philadelphia, Pennsylvania 19122.
D001482:Base Composition; D004256:DNA Polymerase I / Q000378:metabolism*; D004261:DNA Replication; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism; D007700:Kinetics; D008433:Mathematics; D008954:Models, Biological; D011744:Pyrimidinones / Q000378:metabolism*; D013698:Templates, Genetic; D013866:Thioguanine / Q000378:metabolism*; D013942:Thymine Nucleotides / Q000378:metabolism
D003838:Deoxyadenine Nucleotides; D003848:Deoxyguanine Nucleotides; D011744:Pyrimidinones; D013942:Thymine Nucleotides; C056905:5-methyl-2-pyrimidinone; C029603:deoxyguanosine triphosphate; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D013866:Thioguanine; C026600:2'-deoxyadenosine triphosphate; C024157:thymidine 5'-triphosphate