Mace D C
J Biol Chem. 1984 Mar 25;259(6):3616-9.
The incorporation of m6dATP by T4 DNA polymerase has been investigated. Unlike Escherichia coli DNA polymerase I (Engel, J.D., and von Hippel, P.H. (1978) J. Biol. Chem, 253, 935-939), the T4 enzyme discriminates at the insertion step against the methylated triphosphate as compared to the normal substrate (dATP). The apparent Km values measured in two ways agree with the overall 7-fold discrimination measured in double label experiments. The apparent Vmax values measured for net DNA synthesis are the same, while those measured for nucleotide turnover show that the rate for m6dATP is 2-fold greater than for dATP itself. The T4 enzyme results are consistent with the generally held theory that fidelity at the insertion step of DNA polymerization is determined by the relative free energies of primer-enzyme-triphosphate ternary complexes formed by competing, alternative substrate dNTPs. These results are also consistent with the view that these free energies chiefly depend on formation of satisfactory hydrogen bonds between the bases of the template and triphosphate.
人们已经对T4 DNA聚合酶掺入m6dATP的情况进行了研究。与大肠杆菌DNA聚合酶I不同（恩格尔，J.D.，和冯·希佩尔，P.H.（1978年）《生物化学杂志》，253，935 - 939），与正常底物（dATP）相比，T4酶在插入步骤对甲基化三磷酸进行区分。通过两种方式测得的表观Km值与在双标记实验中测得的总体7倍区分度一致。净DNA合成测得的表观Vmax值相同，而核苷酸周转测得的表观Vmax值表明，m6dATP的速率比dATP本身高2倍。T4酶的结果与普遍持有的理论一致，即DNA聚合插入步骤的保真度由竞争的替代底物dNTP形成的引物 - 酶 - 三磷酸三元复合物的相对自由能决定。这些结果也与以下观点一致，即这些自由能主要取决于模板碱基与三磷酸之间形成令人满意的氢键。
