小牛胸腺DNA聚合酶α、δ和ε的生化及功能比较

Biochemical and functional comparison of DNA polymerases alpha, delta, and epsilon from calf thymus.

作者信息

Weiser T, Gassmann M, Thömmes P, Ferrari E, Hafkemeyer P, Hübscher U

机构信息

Department of Pharmacology and Biochemistry, University of Zürich-Irchel, Switzerland.

出版信息

J Biol Chem. 1991 Jun 5;266(16):10420-8.

PMID:1674744

Abstract

DNA polymerase alpha, delta and epsilon can be isolated simultaneously from calf thymus. DNA polymerase delta was purified to apparent homogeneity by a four-column procedure including DEAE-Sephacel, phenyl-Sepharose, phosphocellulose, and hydroxylapatite, yielding two polypeptides of 125 and 48 kDa, respectively. On hydroxylapatite DNA polymerase delta can completely be separated from DNA polymerase epsilon. By KCl DNA polymerase delta is eluted first, while addition of potassium phosphate elutes DNA polymerase epsilon. DNA polymerases delta and epsilon could be distinguished from DNA polymerase alpha by their (i) resistance to the monoclonal antibody SJK 132-20, (ii) relative resistance to N2-[p-(n-butyl)phenyl]-2-deoxyguanosine triphosphate and 2-[p-(n-butyl)anilino]-2-deoxyadenosine triphosphate, (iii) presence of a 3'----5' exonuclease, (iv) polypeptide composition, (v) template requirements, (vi) processivities on the homopolymer poly(dA)/oligo(dT12-18), and (vii) lack of primase. The following differences of DNA polymerase delta to DNA polymerase epsilon were evident: (i) the independence of DNA polymerase epsilon to proliferating cell nuclear antigen for processivity, (ii) utilization of deoxy- and ribonucleotide primers, (iii) template requirements in the absence of proliferating cell nuclear antigen, (iv) mode of elution from hydroxylapatite, and (v) sensitivity to d2TTP and to dimethyl sulfoxide. Both enzymes contain a 3'----5' exonuclease, but are devoid of endonuclease, RNase H, DNA helicase, DNA dependent ATPase, DNA primase, and poly(ADP-ribose) polymerase. DNA polymerase delta is 100-150 fold dependent on proliferating cell nuclear antigen for activity and processivity on poly(dA)/oligo(dT12-18) at base ratios between 1:1 to 100:1. The activity of DNA polymerase delta requires an acidic pH of 6.5 and is also found on poly(dT)/oligo(dA12-18) and on poly(dT)/oligo(A12-18) but not on 10 other templates tested. All three DNA polymerases can be classified according to the revised nomenclature for eukaryotic DNA polymerases (Burgers, P.M. J., Bambara, R. A., Campbell, J. L., Chang, L. M. S., Downey, K. M., Hübscher, U., Lee, M. Y. W. T., Linn, S. M., So, A. G., and Spadari, S. (1990) Eur. J. Biochem. 191, 617-618).

摘要

DNA聚合酶α、δ和ε可从小牛胸腺中同时分离出来。通过包括DEAE-琼脂糖凝胶、苯基琼脂糖凝胶、磷酸纤维素和羟基磷灰石的四步柱层析法，将DNA聚合酶δ纯化至表观均一，分别得到两条分子量为125 kDa和48 kDa的多肽。在羟基磷灰石上，DNA聚合酶δ可与DNA聚合酶ε完全分离。通过KCl可首先洗脱DNA聚合酶δ，而添加磷酸钾则可洗脱DNA聚合酶ε。DNA聚合酶δ和ε可通过以下方面与DNA聚合酶α区分开来：(i) 对单克隆抗体SJK 132-20的抗性；(ii) 对N2-[对-(正丁基)苯基]-2-脱氧鸟苷三磷酸和2-[对-(正丁基)苯胺基]-2-脱氧腺苷三磷酸的相对抗性；(iii) 存在3'→5'外切核酸酶；(iv) 多肽组成；(v) 模板需求；(vi) 对均聚物聚(dA)/寡聚(dT12-18)的持续合成能力；以及(vii) 缺乏引发酶。DNA聚合酶δ与DNA聚合酶ε的以下差异很明显：(i) DNA聚合酶ε的持续合成能力对增殖细胞核抗原的独立性；(ii) 对脱氧核苷酸和核糖核苷酸引物的利用；(iii) 在不存在增殖细胞核抗原时的模板需求；(iv) 从羟基磷灰石上的洗脱方式；以及(v) 对d2TTP和二甲基亚砜的敏感性。这两种酶都含有3'→5'外切核酸酶，但缺乏内切核酸酶、RNase H、DNA解旋酶、依赖DNA的ATP酶、DNA引发酶和聚(ADP-核糖)聚合酶。在碱基比为1:1至100:1时，DNA聚合酶δ在聚(dA)/寡聚(dT12-18)上的活性和持续合成能力对增殖细胞核抗原的依赖性为100-150倍。DNA聚合酶δ的活性需要酸性pH 6.5，并且在聚(dT)/寡聚(dA12-18)和聚(dT)/寡聚(A12-18)上也有活性，但在其他10种测试模板上没有活性。根据真核生物DNA聚合酶的修订命名法（Burgers, P.M. J., Bambara, R. A., Campbell, J. L., Chang, L. M. S., Downey, K. M., Hübscher, U., Lee, M. Y. W. T., Linn, S. M., So, A. G., and Spadari, S. (1990) Eur. J. Biochem. 191, 617-618），所有这三种DNA聚合酶都可进行分类。