小牛胸腺中多种DNA聚合酶α的聚（rA）·寡聚（dT）依赖性活性的进一步表征。

Further characterization of a poly(rA) . oligo(dT)-dependent activity of multiple DNA polymerase alpha from calf thymus.

作者信息

Yoshida S, Masaki S, Koiwai O

出版信息

Biochim Biophys Acta. 1981 Jul 27;654(2):194-200. doi: 10.1016/0005-2787(81)90172-6.

Abstract

DNA polymerase alpha (EC 2.7.7.7) from calf thymus has been separated into three molecular species, i.e., 10 S DNA polymerase alpha, 6.5 S DNA polymerase alpha-1 and 6.5 S DNA polymerase alpha-2 (Masaki, S. and Yoshida, S. (1978) Biochim, Biophys. Acta 531, 74-88; Yoshida, S., Yamada, M., Masaki S. and Seneyoshi, M. (1979) Cancer Res. 39, 3955-3958). Among these three, 10 S DNA polymerase alpha and 6.5 S DNA polymerase alpha-2 were found to copy efficiently poly(rA) . oligo(dT), a template-primer, which was thought to be specific for DNA polymerase gamma or beta. 6.5 S DNA polymerase alpha-1, however, could not use the ribopolymer as a template. The poly(rA) . oligo(dT)-dependent activities of DNA polymerase alpha species differed markedly from those with activated calf thymus DNA in sensitivity to various reagents: the former was inhibited more than 80% by 80 mM KCl, while the latter was stimulated somewhat. Furthermore, aphidicolin, a specific inhibitor of DNA polymerase alpha, did not inhibit the poly(rA) . oligo(dT)-dependent activity. 2',3'-DideoxyTTP, a potent inhibitor of DNA polymerase beta or gamma, slightly inhibited the reactions with poly(rA) . oligo(dT), while it did not inhibit the reactions with activated DNA. The apparent Km values for dTTP on poly(rA) . oligo(dT) template were 260 and 70 microM for 10 S alpha and 6.5 S alpha-2, respectively; these values were much higher than those obtained on activated DNA template (8-10 microM).

摘要

从小牛胸腺中分离出的DNA聚合酶α（EC 2.7.7.7）已被分为三种分子类型，即10S DNA聚合酶α、6.5S DNA聚合酶α-1和6.5S DNA聚合酶α-2（正树，S.和吉田，S.（1978年）《生物化学与生物物理学学报》531，74 - 88；吉田，S.、山田，M.、正树，S.和濑能义，M.（1979年）《癌症研究》39，3955 - 3958）。在这三种类型中，发现10S DNA聚合酶α和6.5S DNA聚合酶α-2能够高效复制聚（rA）·寡聚（dT），这是一种模板引物，曾被认为是DNA聚合酶γ或β所特有的。然而，6.5S DNA聚合酶α-1不能将这种核糖聚合物用作模板。DNA聚合酶α各类型对聚（rA）·寡聚（dT）的依赖性活性在对各种试剂的敏感性方面与对活化小牛胸腺DNA的依赖性活性明显不同：前者在80 mM KCl存在下被抑制超过80%，而后者则受到一定程度的刺激。此外，阿非科林，一种DNA聚合酶α的特异性抑制剂，并不抑制对聚（rA）·寡聚（dT）的依赖性活性。2',3'-双脱氧TTP，一种对DNA聚合酶β或γ有效的抑制剂，对与聚（rA）·寡聚（dT）的反应有轻微抑制作用，而对与活化DNA的反应则无抑制作用。对于聚（rA）·寡聚（dT）模板上的dTTP，10S α和6.5S α-2的表观Km值分别为260和70 μM；这些值远高于在活化DNA模板上获得的值（8 - 10 μM）。