Crute J J, Wahl A F, Bambara R A
Biochemistry. 1986 Jan 14;25(1):26-36. doi: 10.1021/bi00349a005.
Two high molecular weight DNA polymerases, which we have designated delta I and delta II, have been purified from calf thymus tissue. Using Bio Rex-70, DEAE-Sephadex A-25, and DNA affinity resin chromatography followed by sucrose gradient sedimentation, we purified DNA polymerase delta I 1400-fold to a specific activity of 10 000 nmol of nucleotide incorporated h-1 mg-1, and DNA polymerase delta II was purified 4100-fold to a final specific activity of 30 000 nmol of nucleotide incorporated h-1 mg-1. The native molecular weights of DNA polymerase delta I and DNA polymerase delta II are 240 000 and 290 000, respectively. Both enzymes have similarities to other purified delta-polymerases previously reported in their ability to degrade single-stranded DNA in a 3' to 5' direction, affinity for an AMP-hexane-agarose matrix, high activity on poly(dA) X oligo(dT) template, and relative resistance to the polymerase alpha inhibitors N2-(p-n-butylphenyl)dATP and N2-(p-n-butylphenyl)dGTP. These two forms of DNA polymerase delta also share several common features with alpha-type DNA polymerases. Both calf DNA polymerase delta I and DNA polymerase delta II are similar to calf DNA polymerase alpha in molecular weight, are inhibited by the alpha-polymerase inhibitors N-ethylmaleimide and aphidicolin, contain an active DNA-dependent RNA polymerase or primase activity, display a similar extent of processive DNA synthesis, and are stimulated by millimolar concentrations of ATP. We propose that calf DNA polymerase delta I, which also has a template specificity essentially identical with that of calf DNA polymerase alpha, could be an exonuclease-containing form of a DNA replicative enzyme.
我们从小牛胸腺组织中纯化出了两种高分子量DNA聚合酶，分别命名为δI和δII。通过使用Bio Rex - 70、DEAE - Sephadex A - 25和DNA亲和树脂色谱，随后进行蔗糖梯度沉降，我们将DNA聚合酶δI纯化了1400倍，比活性达到每小时每毫克掺入10000纳摩尔核苷酸，DNA聚合酶δII纯化了4100倍，最终比活性为每小时每毫克掺入30000纳摩尔核苷酸。DNA聚合酶δI和DNA聚合酶δII的天然分子量分别为240000和290000。这两种酶在以下方面与先前报道的其他纯化的δ聚合酶相似：能够沿3'至5'方向降解单链DNA、对AMP - 己烷 - 琼脂糖基质有亲和力、在聚（dA）×寡聚（dT）模板上具有高活性以及对聚合酶α抑制剂N2 - （对正丁基苯基）dATP和N2 - （对正丁基苯基）dGTP具有相对抗性。这两种形式的DNA聚合酶δ也与α型DNA聚合酶有几个共同特征。小牛DNA聚合酶δI和DNA聚合酶δII在分子量上与小牛DNA聚合酶α相似，受到α聚合酶抑制剂N - 乙基马来酰亚胺和阿非迪霉素的抑制，含有活性DNA依赖性RNA聚合酶或引发酶活性，显示出相似程度的持续DNA合成，并且受到毫摩尔浓度ATP的刺激。我们提出，小牛DNA聚合酶δI，其模板特异性与小牛DNA聚合酶α基本相同，可能是一种含核酸外切酶形式的DNA复制酶。
