Abdus Sattar A K, Lin T C, Jones C, Konigsberg W H
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520-8024, USA.
Biochemistry. 1996 Dec 24;35(51):16621-9. doi: 10.1021/bi961552q.
Three groups of T4 DNA polymerase mutants were prepared and characterized. In the first group, Ala and Asn were substituted for four acidic residues in the exonuclease domain that were chosen on the basis of their sequence alignment with the Klenow fragment from Escherichia coli DNA polymerase I. Two divalent metal ions required for catalyzing the 3'-5' exonuclease reaction are ligated by carboxyl groups from these conserved Asp and Glu residues. The Ala and Asn replacements have a profound effect on the exonuclease activity of T4 DNA polymerase and also have a significant, but less pronounced influence on its polymerase activity which is located in a domain distal to the exonuclease region. The kcat values for the exonuclease reaction were reduced by 3-4 orders of magnitude by these replacements, but the values of Km(app) did not differ greatly from the wild-type enzyme. The second group consists of replacements of other residues, that are conserved in the exonuclease domain of eukaryotic DNA polymerases, but do not contribute to divalent metal ion coordination. Many of these alterations resulted in decreased exonuclease and/or polymerase activity. Mutants in the third group have substitutions of conserved residues in the polymerase domain which diminished polymerase and altered exonuclease activities. Our results, combined with structural data on crystals of protein N388, a truncated form of T4 DNA polymerase (Wang et al., 1996), show that: (i) the reduction in the relative specific exonuclease activities of mutants in the first group was significantly less than that of mutants in the Klenow fragment, despite the nearly identical geometric arrangement of the metal liganding groups in two proteins; (ii) altered residues, that affect exonuclease and/or polymerase activities in mutants of the second group, cluster within a small area of the exonuclease domain, suggesting that this area may be directly or indirectly involved in polymerase activity; (iii) mutations in the third group, which affect polymerase and exonuclease activities, may participate in DNA and dNTP binding. Our results point to the functional interdependence of the polymerase and exonuclease domains in T4 DNA polymerase, a property not observed with the Klenow fragment.
制备并表征了三组T4 DNA聚合酶突变体。在第一组中，丙氨酸（Ala）和天冬酰胺（Asn）取代了核酸外切酶结构域中的四个酸性残基，这些残基是根据它们与大肠杆菌DNA聚合酶I的Klenow片段的序列比对而选择的。催化3'-5'核酸外切酶反应所需的两个二价金属离子由这些保守的天冬氨酸（Asp）和谷氨酸（Glu）残基的羧基连接。丙氨酸和天冬酰胺取代对T4 DNA聚合酶的核酸外切酶活性有深远影响，对其位于核酸外切酶区域远端结构域的聚合酶活性也有显著但不太明显的影响。这些取代使核酸外切酶反应的kcat值降低了3 - 4个数量级，但Km（app）值与野生型酶没有太大差异。第二组由其他残基的取代组成，这些残基在真核DNA聚合酶的核酸外切酶结构域中保守，但不参与二价金属离子配位。这些改变中的许多导致核酸外切酶和/或聚合酶活性降低。第三组突变体在聚合酶结构域中有保守残基的取代，这降低了聚合酶活性并改变了核酸外切酶活性。我们的结果与T4 DNA聚合酶截短形式蛋白质N388晶体的结构数据（Wang等人，1996）相结合，表明：（i）尽管两种蛋白质中金属配位基团的几何排列几乎相同，但第一组突变体相对特异性核酸外切酶活性的降低明显小于Klenow片段中的突变体；（ii）影响第二组突变体核酸外切酶和/或聚合酶活性的改变残基聚集在核酸外切酶结构域的一个小区域内，表明该区域可能直接或间接参与聚合酶活性；（iii）影响聚合酶和核酸外切酶活性的第三组突变可能参与DNA和dNTP结合。我们的结果表明T4 DNA聚合酶中聚合酶和核酸外切酶结构域的功能相互依存，这是Klenow片段未观察到的特性。
