海拉细胞DNA聚合酶γ：该酶的进一步纯化及特性

HeLa cell DNA polymerase gamma: further purification and properties of the enzyme.

作者信息

Knopf K W, Yamada M, Weissbach A

出版信息

Biochemistry. 1976 Oct 5;15(20):4540-8. doi: 10.1021/bi00665a032.

Abstract

DNA polymerase gamma has been purified over 60 000-fold from HeLa cells which contain no detectable type C viral particles. This purified enzyme shows a specific activity of 25 000 units/mg of protein which is comparable to the known specific activity of homogeneous preparations of human alpha and beta polymerases. The isolated enzyme shows apparent molecular weights ranging from 160 000 to 330 000 according to the method of analysis. The enzyme exhibits optimal activity for copying poly(A) in the presence of 50 mM KPO4 and 130 mM KCl and, under these conditions, copies poly(A) 20 times more rapidly than activated DNA. These assay conditions permit a clear distinction between the gamma-polymerase and DNA polymerase beta which is markedly inhibited by phosphate at this concentration. A comparison of the copying of activated DNA, poly(dA) and poly(A) by DNA polymerases alpha, beta, and gamma under optimal assay conditions for each enzyme is presented. Studies with synthetic and natural nucleic acid templates also show the gamma-polymerase to behave differently that the reverse transcriptases of avian myeloblastosis virus or Rauscher leukemia virus.

摘要

已从不含可检测到的C型病毒颗粒的HeLa细胞中纯化出DNA聚合酶γ，纯化倍数超过60000倍。这种纯化后的酶的比活性为25000单位/毫克蛋白质，这与已知的人α和β聚合酶纯化物的比活性相当。根据分析方法，分离出的酶的表观分子量在160000至330000之间。该酶在50 mM KPO4和130 mM KCl存在下复制聚腺苷酸（poly(A)）时表现出最佳活性，在这些条件下，复制poly(A)的速度比活化DNA快20倍。这些测定条件能够清楚地区分γ聚合酶和DNA聚合酶β，后者在此浓度的磷酸盐存在下会受到显著抑制。本文呈现了在每种酶的最佳测定条件下，DNA聚合酶α、β和γ对活化DNA、聚脱氧腺苷酸（poly(dA)）和poly(A)的复制情况比较。对合成和天然核酸模板的研究还表明，γ聚合酶的行为与禽成髓细胞瘤病毒或劳斯氏白血病病毒的逆转录酶不同。