Kuchta R D, Benkovic P, Benkovic S J
Department of Chemistry, Pennsylvania State University, University Park 16802.
Biochemistry. 1988 Sep 6;27(18):6716-25. doi: 10.1021/bi00418a012.
A complete kinetic scheme describing the polymerization of correct and incorrect dNTPs by the Klenow fragment (KF) of DNA polymerase I has been developed by using short DNA oligomers of defined sequence. The high fidelity arises from a three-stage mechanism. The first stage of discrimination [(1.1 X 10(4-) greater than 1.2 X 10(6]-fold] comes primarily from a dramatically reduced rate of phosphodiester bond formation for incorrect nucleotides, but it also gains a smaller contribution from selective dNTP binding. After phosphodiester bond formation, a conformational change slows dissociation of the incorrect DNA products from KF and, in conjunction with editing by the 3'----5'-exonuclease, increases fidelity 4- greater than 61-fold. Finally, KF polymerizes the next correct dNTP onto a mismatch very slowly, providing a further 6- greater than 340-fold increase in fidelity. Surprisingly, the 3'----5'-exonuclease did not in its hydrolysis reaction differentiate between correctly and incorrectly base-paired nucleotides; rather, an increased lifetime of the enzyme-DNA complex containing the misincorporated base is responsible for discrimination.
通过使用特定序列的短DNA寡聚物，已构建出一个完整的动力学方案，用于描述DNA聚合酶I的Klenow片段（KF）对正确和错误dNTP的聚合作用。高保真度源于一个三阶段机制。第一阶段的辨别作用[（1.1×10⁴至大于1.2×10⁶倍）]主要源于错误核苷酸形成磷酸二酯键的速率大幅降低，但选择性dNTP结合也有较小贡献。磷酸二酯键形成后，构象变化减缓了错误DNA产物从KF上的解离，并与3'→5'外切核酸酶的编辑作用相结合，使保真度提高4至大于61倍。最后，KF将下一个正确的dNTP聚合到错配上的速度非常慢，使保真度进一步提高6至大于340倍。令人惊讶的是，3'→5'外切核酸酶在其水解反应中并未区分正确和错误碱基配对的核苷酸；相反，含有错配碱基的酶-DNA复合物寿命延长才是产生辨别的原因。
