CONSTRUCTION AND CHARACTERIZATION OF A BACTERIOPHAGE-T4 DNA-POLYMERASE DEFICIENT IN 3'-]5' EXONUCLEASE ACTIVITY
CONSTRUCTION AND CHARACTERIZATION OF A BACTERIOPHAGE-T4 DNA-POLYMERASE DEFICIENT IN 3'-]5' EXONUCLEASE ACTIVITY
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DOI：
10.1073/pnas.90.7.2579
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发表时间：
1993-04-01
影响因子：
11.1
通讯作者：
BENKOVIC, SJ
中科院分区：
文献类型：
--
作者：
FREY, MW;NOSSAL, NG;BENKOVIC, SJ
噬菌体T4 DNA聚合酶具有校对3‘ -> 5’外切酶，对维持DNA复制的准确性起重要作用。我们通过将Asp-219转化为Ala构建了一个T4 DNA聚合酶缺失的核酸外切酶。突变体T4 DNA聚合酶的外切酶活性降低了至少10倍(7)，但它保留了聚合酶活性，其动力学参数k（cat）、k(d) DNA和k(d) dATP与野生型酶非常接近。携带突变聚合酶基因的T4噬菌体突变频率显著增加。T4 DNA聚合酶中的Asp-219与先前证明的大肠杆菌DNA聚合酶I （Asp-424）、噬菌体phi29 DNA聚合酶（Asp-66）和酵母DNA聚合酶delta （Asp-405）的Klenow片段的核酸外切酶活性所必需的Asp残基周围的序列相似。因此，这些研究支持了这些和相关DNA聚合酶的校对外切酶的活性位点存在相似序列的建议。
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, k(cat), K(d) DNA, and K(d) 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 phi29 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.