Bebenek K, Joyce C M, Fitzgerald M P, Kunkel T A
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
J Biol Chem. 1990 Aug 15;265(23):13878-87.
The fidelity of DNA synthesis by an exonuclease-proficient DNA polymerase results from the selectivity of the polymerization reaction and from exonucleolytic proofreading. We have examined the contribution of these two steps to the fidelity of DNA synthesis catalyzed by the large Klenow fragment of Escherichia coli DNA polymerase I, using enzymes engineered by site-directed mutagenesis to inactivate the proofreading exonuclease. Measurements with two mutant Klenow polymerases lacking exonuclease activity but retaining normal polymerase activity and protein structure demonstrate that the base substitution fidelity of polymerization averages one error for each 10,000 to 40,000 bases polymerized, and can vary more than 30-fold depending on the mispair and its position. Steady-state enzyme kinetic measurements of selectivity at the initial insertion step by the exonuclease-deficient polymerase demonstrate differences in both the Km and the Vmax for incorrect versus correct nucleotides. Exonucleolytic proofreading by the wild-type enzyme improves the average base substitution fidelity by 4- to 7-fold, reflecting efficient proofreading of some mispairs and less efficient proofreading of others. The wild-type polymerase is highly accurate for -1 base frameshift errors, with an error rate of less than or equal to 10(-6). The exonuclease-deficient polymerase is less accurate, suggesting that proofreading also enhances frameshift fidelity. Even without a proofreading exonuclease, Klenow polymerase has high frameshift fidelity relative to several other DNA polymerases, including eucaryotic DNA polymerase-alpha, an exonuclease-deficient, 4-subunit complex whose catalytic subunit is almost three times larger. The Klenow polymerase has a large (46 kDa) domain containing the polymerase active site and a smaller (22 kDa) domain containing the active site for the 3'----5' exonuclease. Upon removal of the small domain, the large polymerase domain has altered base substitution error specificity when compared to the two-domain but exonuclease-deficient enzyme. It is also less accurate for -1 base errors at reiterated template nucleotides and for a 276-nucleotide deletion error. Thus, removal of a protein domain of a DNA polymerase can affect its fidelity.
具有核酸外切酶活性的DNA聚合酶进行DNA合成时的保真度，源于聚合反应的选择性以及核酸外切酶校对机制。我们利用定点诱变技术构建了缺乏校对核酸外切酶活性的工程酶，研究了这两个步骤对大肠杆菌DNA聚合酶I的大片段Klenow催化DNA合成保真度的贡献。对两种缺乏核酸外切酶活性但保留正常聚合酶活性和蛋白质结构的突变型Klenow聚合酶进行测量，结果表明，聚合反应的碱基替换保真度平均为每聚合10000至40000个碱基出现一次错误，并可能因错配及其位置的不同而相差30倍以上。对缺乏核酸外切酶的聚合酶在初始插入步骤的选择性进行稳态酶动力学测量，结果显示，对于错误和正确的核苷酸，Km和Vmax均存在差异。野生型酶的核酸外切酶校对功能使平均碱基替换保真度提高了4至7倍，这反映出对某些错配的校对效率较高，而对其他错配的校对效率较低。野生型聚合酶在-1碱基移码错误方面具有高度准确性，错误率小于或等于10^(-6)。缺乏核酸外切酶的聚合酶准确性较低，这表明校对功能也提高了移码保真度。即使没有校对核酸外切酶，相对于其他几种DNA聚合酶，包括真核生物DNA聚合酶α（一种缺乏核酸外切酶的四亚基复合物，其催化亚基几乎大三倍），Klenow聚合酶仍具有较高的移码保真度。Klenow聚合酶有一个较大的结构域（46 kDa），其中包含聚合酶活性位点，还有一个较小的结构域（22 kDa），其中包含3'→5'核酸外切酶的活性位点。去除小结构域后，与双结构域但缺乏核酸外切酶的酶相比，大聚合酶结构域的碱基替换错误特异性发生了改变。在重复模板核苷酸处发生-1碱基错误以及出现276个核苷酸缺失错误时，其准确性也较低。因此，去除DNA聚合酶的一个蛋白质结构域会影响其保真度。
