Longley M J, Mosbaugh D W
Department of Agricultural Chemistry, Oregon State University, Corvallis 97331.
J Biol Chem. 1991 Dec 25;266(36):24702-11.
Porcine liver DNA polymerase gamma was shown previously to copurify with an associated 3' to 5' exonuclease activity (Kunkel, T. A., and Mosbaugh, D. W. (1989) Biochemistry 28, 988-995). The 3' to 5' exonuclease has now been characterized, and like the DNA polymerase activity, it has an absolute requirement for a divalent metal cation (Mg2+ or Mn2+), a relatively high NaCl and KCl optimum (150-200 mM), and an alkaline pH optimum between 7 and 10. The exonuclease has a 7.5-fold preference for single-stranded over double-stranded DNA, but it cannot excise 3'-terminal dideoxy-NMP residues from either substrate. Excision of 3'-terminally mismatched nucleotides was preferred approximately 5-fold over matched 3' termini, and the hydrolysis product from both was a deoxyribonucleoside 5'-monophosphate. The kinetics of 3'-terminal excision were measured at a single site on M13mp2 DNA for each of the 16 possible matched and mismatched primer.template combinations. As defined by the substrate specificity constant (Vmax/Km), each of the 12 mismatched substrates was preferred over the four matched substrates (A.T, T.A, C.G, G.C). Furthermore, the exonuclease could efficiently excise internally mismatched nucleotides up to 4 residues from the 3' end. DNA polymerase gamma was not found to possess detectable DNA primase, endonuclease, 5' to 3' exonuclease, RNase, or RNase H activities. The DNA polymerase and exonuclease activities exhibited dissimilar rates of heat inactivation and sensitivity to N-ethylmaleimide. After nondenaturing activity gel electrophoresis, the DNA polymerase and 3' to 5' exonuclease activities were partially resolved and detected in situ as separate species. A similar analysis on a denaturing activity gel identified catalytic polypeptides with molecular weights of 127,000, 60,000, and 32,000 which possessed only DNA polymerase gamma activity. Collectively, these results suggest that the polymerase and exonuclease activities reside in separate polypeptides, which could be derived from separate gene products or from proteolysis of a single gene product.
先前已表明，猪肝DNA聚合酶γ能与一种相关的3'至5'核酸外切酶活性共纯化（Kunkel, T. A., and Mosbaugh, D. W. (1989) Biochemistry 28, 988 - 995）。现在已对该3'至5'核酸外切酶进行了特性分析，与DNA聚合酶活性一样，它对二价金属阳离子（Mg2+或Mn2+）有绝对需求，NaCl和KCl的最适浓度相对较高（150 - 200 mM），最适碱性pH在7至10之间。该核酸外切酶对单链DNA的偏好性比对双链DNA高7.5倍，但它不能从任何一种底物上切除3'末端的双脱氧-NMP残基。切除3'末端错配核苷酸的偏好性比对匹配的3'末端约高5倍，两者的水解产物均为脱氧核糖核苷5'-单磷酸。针对M13mp2 DNA上的单个位点，对16种可能的匹配和错配引物-模板组合分别测定了3'末端切除的动力学。根据底物特异性常数（Vmax/Km）定义，12种错配底物中的每一种都比4种匹配底物（A.T、T.A、C.G、G.C）更受偏好。此外，该核酸外切酶能有效切除3'末端内部错配的核苷酸，距离3'端最远可达4个残基。未发现DNA聚合酶γ具有可检测到的DNA引发酶、内切酶、5'至3'核酸外切酶、RNase或RNase H活性。DNA聚合酶和核酸外切酶活性表现出不同的热失活速率以及对N-乙基马来酰亚胺的敏感性。在非变性活性凝胶电泳后，DNA聚合酶和3'至5'核酸外切酶活性部分分离，并原位检测为不同的物种。在变性活性凝胶上进行的类似分析鉴定出分子量分别为127,000、60,000和32,000的催化多肽，它们仅具有DNA聚合酶γ活性。总体而言，这些结果表明聚合酶和核酸外切酶活性存在于不同的多肽中，这可能来自不同的基因产物或单个基因产物的蛋白水解。
