Reha-Krantz L J, Nonay R L
Department of Genetics, University of Alberta, Edmonton, Canada.
J Biol Chem. 1993 Dec 25;268(36):27100-8.
DNA polymerase exonucleolytic proofreading is important in attaining high fidelity DNA replication. One of the most well characterized proofreading activities is the 3'-->5'-exonuclease activity of bacteriophage T4 DNA polymerase. We have used genetic analyses and protein sequence comparisons to Escherichia coli DNA polymerase I to identify amino acids in the N-terminal region of T4 DNA polymerase that are required for exonucleolytic proofreading. Mutant DNA polymerases with amino acid substitutions D112A/E114A, D219A, or D324A reduced 3'-->5'-exonuclease activity 10(2)-10(4)-fold in various in vitro assays and decreased DNA replication fidelity in vivo. DNA replication activity was also reduced for the exonuclease-deficient DNA polymerases in vitro and in vivo. Reduction in DNA replication appeared to be due primarily to the interdependence of T4 DNA polymerase replication and proofreading activities; T4 DNA polymerase requires 3'-->5'-exonuclease activity to repair primer termini that are not suitable substrates for extension. Observations reported here provide further evidence in support of the proposal that DNA polymerases have distinct 3'-->5'-exonuclease and polymerase active sites.
DNA聚合酶的核酸外切酶校对功能对于实现高保真度的DNA复制至关重要。最具特征的校对活性之一是噬菌体T4 DNA聚合酶的3'→5'-核酸外切酶活性。我们利用对大肠杆菌DNA聚合酶I的遗传分析和蛋白质序列比较，来确定T4 DNA聚合酶N端区域中核酸外切酶校对所需的氨基酸。在各种体外试验中，氨基酸取代为D112A/E114A、D219A或D324A的突变DNA聚合酶使3'→5'-核酸外切酶活性降低了10²-10⁴倍，并在体内降低了DNA复制保真度。体外和体内实验中，核酸外切酶缺陷型DNA聚合酶的DNA复制活性也有所降低。DNA复制的减少似乎主要是由于T4 DNA聚合酶复制和校对活性之间的相互依赖性；T4 DNA聚合酶需要3'→5'-核酸外切酶活性来修复那些不适于延伸的引物末端。本文报道的观察结果为DNA聚合酶具有不同的3'→5'-核酸外切酶和聚合酶活性位点这一观点提供了进一步的证据。
