Frey M W, Nossal N G, Capson T L, Benkovic S J
Pennsylvania State University, Department of Chemistry, Davey Laboratory, University Park 16802.
Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2579-83. doi: 10.1073/pnas.90.7.2579.
Bacteriophage T4 DNA polymerase has a proofreading 3'-->5' exonuclease that plays an important role in maintaining the accuracy of DNA replication. We have constructed a T4 DNA polymerase deficient in this exonuclease by converting Asp-219 to Ala. The exonuclease activity of the mutant T4 DNA polymerase has been reduced by a factor of at least 10(7), but it retains a polymerase activity whose kinetic parameters, kcat, Kd DNA, and Kd dATP, are very close to those of the wild-type enzyme. Bacteriophage T4 with the mutant polymerase gene has a markedly increased mutation frequency. Asp-219 in T4 DNA polymerase is within a sequence similar to those surrounding Asp residues previously shown to be essential for the exonuclease activities of the Klenow fragment of Escherichia coli DNA polymerase I (Asp-424), bacteriophage phi 29 DNA polymerase (Asp-66), and Saccharomyces cerevisiae DNA polymerase delta (Asp-405). Thus, these studies support the proposal that there are similar sequences in the active sites for the proofreading exonucleases of these and related DNA polymerases.
噬菌体T4 DNA聚合酶具有一个校正3'→5'核酸外切酶，该酶在维持DNA复制的准确性方面发挥着重要作用。我们通过将天冬氨酸-219转换为丙氨酸，构建了一种缺乏这种核酸外切酶的T4 DNA聚合酶。突变型T4 DNA聚合酶的核酸外切酶活性至少降低了10^7倍，但它保留了一种聚合酶活性，其动力学参数kcat、Kd DNA和Kd dATP与野生型酶非常接近。携带突变聚合酶基因的噬菌体T4具有明显增加的突变频率。T4 DNA聚合酶中的天冬氨酸-219位于一个与先前显示对大肠杆菌DNA聚合酶I的Klenow片段（天冬氨酸-424）、噬菌体φ29 DNA聚合酶（天冬氨酸-66）和酿酒酵母DNA聚合酶δ（天冬氨酸-405）的核酸外切酶活性至关重要的天冬氨酸残基周围的序列相似的序列内。因此，这些研究支持了这样的提议，即这些及相关DNA聚合酶的校正核酸外切酶的活性位点存在相似序列。
