FURTHER-STUDIES ON CALF THYMUS DNA POLYMERASE-DELTA PURIFIED TO HOMOGENEITY BY A NEW PROCEDURE
FURTHER-STUDIES ON CALF THYMUS DNA POLYMERASE-DELTA PURIFIED TO HOMOGENEITY BY A NEW PROCEDURE
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DOI：
10.1021/bi00304a003
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发表时间：
1984-01-01
期刊：
影响因子：
2.9
通讯作者：
SO, AG
中科院分区：
文献类型：
--
作者：
LEE, MYWT;TAN, CK;SO, AG
DNA聚合酶δ。通过一种新程序将来自小牛胸腺的α-淀粉样蛋白纯化至表观同质性，该新程序在早期步骤中利用带有苯基-琼脂糖的疏水相互作用层析来从DNA聚合酶δ中分离大部分Ca依赖性蛋白酶活性。纯化的酶在非变性条件下在聚丙烯酰胺凝胶电泳上作为单一蛋白带迁移。该酶的沉降系数为7.9S，斯托克斯半径为53埃。计算出天然酶的MW为173kDa[千道尔顿]。均质酶的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳揭示了125和48 kDa的2种多肽。该亚基结构不同于DNA聚合酶δ的亚基结构。该多肽由先前的程序制备，由 60 和 49 kDa 的亚基组成，表明 60-kDa 多肽可能是在酶纯化过程中衍生自 125-kDa 多肽，可能是异常敏感肽键裂解的结果。 DNA聚合酶δ。与DNA聚合酶.α分离。通过苯基-琼脂糖凝胶上的疏水相互作用层析； DNA聚合酶δ。在pH 7.2和DNA聚合酶.α下洗脱。 pH 值 8.5。 DNA聚合酶δ。也可以与DNA聚合酶.α分离。通过羟基磷灰石色谱法； DNA聚合酶.α。在 0.5 M KCl 存在的情况下与羟基磷灰石结合，而 DNA 聚合酶 .delta 则与羟基磷灰石结合。在 90 mM KCl 下洗脱。 DNA聚合酶δ。与3''-至5''-核酸外切酶活性相关，但其缺乏核酸内切酶或5''-至3''-核酸外切酶活性。 DNA聚合酶δ的聚合酶活性。当底物是双链 DNA 时，Aphidicolin 会抑制 3''- 至 5''-核酸外切酶活性。 Aphidicolin 不抑制单链 DNA 上的核酸外切酶活性。 DNA聚合酶δ的聚合酶和核酸外切酶活性的协调抑制。阿菲迪霉素的作用与两种活性驻留在同一蛋白质分子上的观察结果一致。目前的发现也与阿菲迪霉素与酶的结合需要形成模板-引物-DNA聚合酶复合物的建议一致。
DNA polymerase .delta. from calf thymus was purified to apparent homogeneity by a new procedure which utilizes hydrophobic interaction chromatography with phenyl-Sepharose at an early step to separate most of the Ca-dependent protease activity from DNA polymerases .delta. and .alpha.. The purified enzyme migrates as a single protein band on polyacrylamide gel electrophoresis under nondenaturing conditions. The sedimentation coefficient of the enzyme is 7.9S, and the Stokes radius is 53 .ANG.. A MW of 173 kDa [kilodaltons] was calculated for the native enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the homogeneous enzyme reveals 2 polypeptides of 125 and 48 kDa. This subunit structure differs from that of DNA polymerase .delta. prepared by a previous procedure, which was composed of subunits of 60 and 49 kDa, suggesting that the 60-kDa polypeptide may have been derived from the 125-kDa polypeptide during enzyme purification, possibly as the result of cleavage of an unusually sensitive peptide bond. DNA polymerase .delta. is separated from DNA polymerase .alpha. by hydrophobic interaction chromatography on phenyl-Sepharose; DNA polymerase .delta. is eluted at pH 7.2 and DNA polymerase .alpha. at pH 8.5. DNA polymerase .delta. can also be separated from DNA polymerase .alpha. by chromatography on hydroxylapatite; DNA polymerase .alpha. binds to hydroxylapatite in the presence of 0.5 M KCl, whereas DNA polymerase .delta. is eluted at 90 mM KCl. DNA polymerase .delta. is associated with a 3''- to 5''-exonuclease activity, but it is devoid of endonuclease or 5''- to 3''-exonuclease activities. The polymerase activity of DNA polymerase .delta. is inhibited by aphidicolin, as is the 3''- to 5''-exonuclease activity when the substrate is double-stranded DNA. Aphidicolin does not inhibit exonuclease activity on single-stranded DNA. The coordinated inhibition of both polymerase and exonuclease activities of DNA polymerase .delta. by aphidicolin is consistent with the observation that both activities reside on the same protein molecule. The present findings are also consistent with the suggestion that the binding of aphidicolin to the enzyme requires the formation of a template-primer-DNA polymerase complex.