Rappaport H P
Biology Department, Temple University, Philadelphia, Pennsylvania 19122.
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.
利用含有大肠杆菌DNA聚合酶I大片段Klenow片段的特定寡聚物，已测定了碱基对6-硫鸟嘌呤（Gs）/5-甲基-2-嘧啶酮（Th）的复制动力学和保真度。Th与Gs配对时的插入效率Vmax/Km（min-1 microM-1）为1.5，Gs与Th配对时的插入效率为0.7。相比之下，C与G配对以及G与C配对时的插入效率分别为0.5和1.5。在Gs/Th碱基对之后，下一个碱基A与T配对时的插入效率比G/C碱基对之后高2倍。就胸腺嘧啶和腺嘌呤而言，复制保真度的错配插入频率，即“错误”碱基与“正确”碱基插入效率之比，T与Gs配对（T/Gs）时为7×10-4，T/Th时为4×10-6，A/Th时最大稳定错配插入频率为4×10-4。当A插入到与Gs相对的位置后，未检测到延伸现象。这些值与G和C与T和A配对时的错配插入频率相似。G和C的最大稳定错配插入频率，G/Th时为4×10-2，Gs/C时为3×10-2 - 7×10-3，C/Gs时为2.6×10-1，Th/G时错配插入频率<1×10-3。动力学结果和分子模型表明，对Gs/Th碱基对进行修饰可能会提高C和G的复制保真度。
