Craig R K, Keir H M
Biochem J. 1975 Feb;145(2):215-24. doi: 10.1042/bj1450215.
DNA polymerase from BHK-21/C13 cells were separated into two species, DNA polymerase I corresponding to the heterogeneous enzyme with sedimentation coefficient of 6-8S, and DNA polymerase II, corresponding to the enzyme with sedimentation coefficient of 3.3S. DNA polymerase I was purified 114-fold and DNA polymerase II 154-fold by a simple extraction procedure followed by column chromatography on phosphocellulose and gel filtration through Sephadex G-100. The purified enzymes differed markedly in respect of pH optimum, stimulation and inhibition by K+, Km for the deoxyribonucleoside 5'-triphosphates, stability to heating at 45 degrees C, and inhibition by N-ethylmaleimide. The preferred primer-template for both enzymes was "activated" DNA (DNA submitted to limited degradation by pancreatic deoxyribonuclease); native or thermally denatured DNA templates were relatively very poorly copied. When certain synthetic templates were tested, substantial differences were revealed between the two enzymes. Poly[d(A-T)] was poorly used by polymerase I but was superior to "activated" DNA for polymerase II. Poly[d(A)]-oligo[d(pT)10] was used efficiently by polymerase I but not by polymerase II. Poly(A)-oligo[d(pT)10] was not an effective primer-template although polymerase I could use it to a limited extent when Mn2+ replaced Mg2+ in the polymerase reaction and when the temperature of incubation was lowered from 37 degrees to 30 degrees C. When only one or two or three triphosphates were supplied in the reaction mixture, the activity of polymerase I was more severly diminished than that of polymerase II.
从BHK - 21/C13细胞中分离出的DNA聚合酶可分为两种：DNA聚合酶I，对应沉降系数为6 - 8S的异质性酶；以及DNA聚合酶II，对应沉降系数为3.3S的酶。通过简单的提取程序，随后进行磷酸纤维素柱色谱和Sephadex G - 100凝胶过滤，DNA聚合酶I被纯化了114倍，DNA聚合酶II被纯化了154倍。纯化后的酶在最适pH、K⁺的刺激和抑制作用、对脱氧核糖核苷5'-三磷酸的Km值、45℃加热稳定性以及N - 乙基马来酰亚胺的抑制作用等方面存在显著差异。两种酶的首选引物模板均为“活化”DNA（经胰脱氧核糖核酸酶有限降解的DNA）；天然或热变性的DNA模板复制效果相对较差。当测试某些合成模板时，两种酶之间显示出显著差异。聚合酶I对聚[d(A - T)]的利用较差，但对聚合酶II而言优于“活化”DNA。聚合酶I能有效利用聚[d(A)] - 寡聚[d(pT)10]，而聚合酶II则不能。聚(A) - 寡聚[d(pT)10]不是有效的引物模板，尽管当在聚合酶反应中用Mn²⁺取代Mg²⁺且孵育温度从37℃降至30℃时，聚合酶I能有限程度地利用它。当反应混合物中仅提供一、二或三种三磷酸时，聚合酶I活性的降低比聚合酶II更严重。
