Resolution and purification of free primase activity from the DNA primase-polymerase alpha complex of HeLa cells.
Resolution and purification of free primase activity from the DNA primase-polymerase alpha complex of HeLa cells.
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从 HeLa 细胞的 DNA 引物酶-聚合酶 α 复合物中分离和纯化游离引物酶活性。
DOI：
10.1093/nar/14.21.8467
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发表时间：
1986
影响因子：
14.9
通讯作者：
Baril,EF
中科院分区：
文献类型：
--
作者：
Vishwanatha,JK;Baril,EF
用琼脂糖亲和层析分离纯化的HeLa细胞DNA引物酶-聚合酶α复合物中的DNA引物酶活性。该方法提供了不含聚合酶α的DNA引发酶的良好产率（55%）。游离的DMA引发酶活性被纯化至接近均一性，并对其性质进行表征。纯化的游离DNA引发酶的十二烷基硫酸钠聚丙烯酰胺凝胶电泳分析显示Mr 70，000的主要蛋白质染色条带。在velcity沉降中的天然酶具有5的S20，W。DNA引发酶以单链M-13 DNA、聚（dT）和聚（dC）模板合成RNA寡聚体，这些模板以几种纯化的真核DNA引发酶-聚合酶α复合物中已描述的方式被DNA聚合酶α延长。纯化的游离DNA打印酶活性对特异性抑制游离DNA聚合酶α以及与引发酶复合的DNA聚合酶α的中和抗人DNA聚合酶α抗体、BuPdGTP和阿非迪霉素具有抗性。游离引发酶活性对单价盐浓度更敏感，并且比聚合酶α更不稳定。总之，这些结果表明DNA引发酶-聚合酶α复合物的DNA引发酶和聚合酶α活性存在于通过疏水相互作用紧密结合的单独多肽上。
DNA primase activlty has been resolved from a purified DNA primase-polymerase α complex of HeLa cells by hydrophobia affinity chromatography on phenylSepharose followed by chromatography on hexylagarose. This procedure provides a good yield (55%) of DNA primase that is free from polymerase α. The free DMA primase activity was purified to near homogeneity and its properties characterized. Sodium dodecyl sulfate polyaorylanide gel electrophoretio analysis of the purified free DNA primase showed a major protein staining band of Mr70,000. The native enzyme in velcity sedimentation has an S20, W of 5. DNA primase synthesizes RNA ollgomers with single- stranded M-13 DNA, poly(dT) and poly(dC) templates that are elongated by the DNA polymerase α in a manner that has already been described for several purified eukaryotic DNA primase-polymerase α complexes. The purified free DNA prinase activity is resistant to neutralizing anti-human DNA polymerase α antibodies, BuPdGTP and aphidicolin that specifically inhibit the free DNA polymerase α and also DNA polymerase α complexed with the primase. The free primase activity is more sensitive to monovalent salt concentrations and is more labile than polymerase α. Taken together these results indicate that the DNA primase and polymerase α activities of the DNA primase-polymerase α complex reside on separate polypeptides that associate tightly through hydrophobio Interactions.