REPLICATION OF THE BASE PAIR 6-THIOGUANINE/5-METHYL-2-PYRIMIDINONE WITH THE LARGE KLENOW FRAGMENT OF ESCHERICHIA-COLI DNA-POLYMERASE-I

The kinetics and the fidelity of replication of the base pair 6-thioguanine (G(s))/5-methyl-2-pyrimidinone (T(h)) have been determined by using defined oligomers with the large Klenow fragment of Escherichia coli DNA polymerase 1. The insertion efficiency, V(max)/K(m) (min-1 muM-1), of T(h) opposite G(s) is 1.5, and the insertion efficiency of G(s) opposite T(h) 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 G(s)/T(h) 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 G(s) (T/G(s)), 4 X 10(-6) for T/T(h), and a maximum stable misinsertion frequency of 4 X 10(-4) for A/T(h). No detectable elongation occurs after an A is inserted opposite a G(s). 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/T(h), 3 X 10(-2)-7 X 10(-3) for G(s)/C, and 2.6 X 10(-1) for C/G(s), and the misinsertion frequency is <1 X 10(-3) for T(h)/G. The kinetics results and molecular modeling suggest modifications to the G(s)/T(h) base pair that may provide higher levels of fidelity of replication with respect to C and G.