Ito K, Arens M, Green M
Biochim Biophys Acta. 1976 Oct 18;447(3):340-52. doi: 10.1016/0005-2787(76)90057-5.
We have isolated a nuclear membrane fraction from KB cells infected with human adenovirus 2 that synthesizes exclusively small viral DNA chains (approx. 9 S) in vitro (Yamashita, T., Arens, M. and Green, M. (1975) J. Biol. Chem. 250, 3273-3279). The DNA polymerase activity present in the adenovirus 2 DNA-nuclear membrane complex was purified through chromatography on phosphocellulose and DEAE-cellulose, glycerol gradient centrifuation and DNA-cellulose chromatography. A single peak of enzymatic activity sedimented in glycerol gradients at about 6.7 S which corresponds to a molecular weight of 125000. The enzyme preparation in the step of glycerol gradient centrifugation utilized activated calf thymus, KB cell and adenovirus 2 DNA as template-primer in the presence of Mg2+; Km values for these DNAs were 5.5, 4.0, and 0.8 mug/ml, respectively. The partially purified enzyme preparation was characterized by several criteria which were compared to the properties of the three major mammalian DNA polymerases, alpha, beta, and psi. On the basis of template-primer preference, effect of salt, inhibition by N-ethylmaleimide and Km for dTTP, the DNA polymerase activity from the membrane complex can be distinguished from the alpha and beta DNA polymerases. The elution profile from DNA cellulose revealed a minor peak (I) and a major peak (II) of DNA polymerase activity utilizing poly(A) -(dT)10 as template-primer in the presence of Mn2+ - Peak II from DNA cellulose, which contained about 90% of the total DNA polymerase activity eluted from the column, was 2-3 times as active with poly(A) - (dT)10 as template-primer in the presence of Mn2+ than with activated calf thymus DNA in the presence of Mg2+. On the other hand, peak I had a low ratio of poly(A) - (dT)10 to activated calf thymus DNA activity. DNA polymerase was also purified from the nuclear membrane fraction of uninfected KB cells by the same procedures as those used in enzyme purification from the adenovirus 2 DNA-nuclear membrane complex. A minor peak and a major peak of DNA polymerase activity utilizing poly(A) - (dT)10 as template primer in the presence of Mn2+ were again observed that eluted from DNA cellulose at the same KCl concentrations as peak I and II from adenovirus 2-infected cells. The enzymes of the nuclear membrane fraction of uninfected KB cells could not be differentiated from the enzymes of the adenovirus 2 DNA-nuclear membrane complex through any of the purification steps nor by their template specificities. These results indicate that the predominant enzyme in the adenovirus 2 DNA-nuclear membrane complex and in the KB cell nuclear membrane complex belongs to the class of DNA polymerase psi.
我们从感染了人腺病毒2的KB细胞中分离出了一种核膜组分，该组分在体外仅合成小的病毒DNA链（约9S）（山下，T.，阿伦斯，M.和格林，M.（1975年）《生物化学杂志》250，3273 - 3279）。腺病毒2 DNA - 核膜复合物中存在的DNA聚合酶活性通过磷酸纤维素和DEAE - 纤维素柱层析、甘油梯度离心以及DNA - 纤维素柱层析进行纯化。在甘油梯度中，酶活性的单一峰在约6.7S处沉降，这对应于分子量为125000。甘油梯度离心步骤中的酶制剂在Mg2 +存在下以活化的小牛胸腺DNA、KB细胞DNA和腺病毒2 DNA作为模板 - 引物；这些DNA的Km值分别为5.5、4.0和0.8μg/ml。部分纯化的酶制剂通过几个标准进行了表征，并与三种主要的哺乳动物DNA聚合酶α、β和ψ的特性进行了比较。基于模板 - 引物偏好、盐的影响、N - 乙基马来酰亚胺的抑制作用以及对dTTP的Km值，膜复合物中的DNA聚合酶活性可以与α和β DNA聚合酶区分开来。在Mn2 +存在下，以聚（A） - （dT）10作为模板 - 引物时，DNA纤维素的洗脱图谱显示出一个小峰（I）和一个大峰（II）的DNA聚合酶活性。来自DNA纤维素的峰II含有从柱上洗脱的总DNA聚合酶活性的约90%，在Mn2 +存在下以聚（A） - （dT）10作为模板 - 引物时的活性是在Mg2 +存在下以活化的小牛胸腺DNA时活性的2 - 3倍。另一方面，峰I的聚（A） - （dT）10与活化的小牛胸腺DNA活性的比率较低。通过与从腺病毒2 DNA - 核膜复合物中纯化酶相同的程序，也从未感染的KB细胞核膜组分中纯化了DNA聚合酶。在Mn2 +存在下，以聚（A） - （dT）10作为模板引物时，再次观察到DNA聚合酶活性的一个小峰和一个大峰，它们在与腺病毒2感染细胞的峰I和峰II相同的KCl浓度下从DNA纤维素中洗脱。在任何纯化步骤中，未感染的KB细胞核膜组分的酶与腺病毒2 DNA - 核膜复合物的酶都无法通过其模板特异性进行区分。这些结果表明，腺病毒2 DNA - 核膜复合物和KB细胞核膜复合物中的主要酶属于DNA聚合酶ψ类别。
