Single-stranded-DNA-dependent ATPase from HeLa cells that stimulates DNA polymerase alpha-primase activity: purification and characterization of the ATPase.
Single-stranded-DNA-dependent ATPase from HeLa cells that stimulates DNA polymerase alpha-primase activity: purification and characterization of the ATPase.
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来自 HeLa 细胞的单链 DNA 依赖性 ATP 酶可刺激 DNA 聚合酶 α-引物酶活性：ATP 酶的纯化和表征。
DOI：
10.1021/bi00489a036
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发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Baril,EF
中科院分区：
文献类型：
--
作者：
Vishwanatha,JK;Baril,EF
1990年6月15日收到的修订手稿摘要：HeLa细胞中的一个多蛋白DNA聚合酶复合体在纯化的早期阶段参与分离的单链DNA依赖的ATPase已被纯化为均一。ATPase是16S多酶DNA聚合酶的一部分，该复合体在体外完全活跃于SV40DNA复制。在一个完全依赖于DNA存在的反应中，ATPase将ATP水解成ADP。单链DNA作为辅因子是首选的，含腺嘌呤或胸苷残基的多脱氧核苷酸是非常有效的。甘油梯度沉淀法表明，纯化后的ATPase经52o，w=7 S沉淀，变性条件下的聚丙烯酰胺凝胶电泳显示两个相对分子质量分别为83000和68000的多肽。光亲和交联实验表明，这两种多肽都具有嘌呤核苷酸结合部位。ATP与这两个亚基的结合比GTP更有效，CTP或UTP不与这两个多肽发生交联反应。纯化的HeLa细胞DNA聚合酶α-Primase催化的DNA合成在ATPase和ATP的存在下被刺激，最适浓度为2 mM。DNA产物的凝胶电泳法分析表明，以聚(DT)而不是噬菌体M13 DNA为模板，ATPase克服了滞后现象，减少了DNA聚合酶a-Primase复合体合成的新生DNA链的长度。
Revised Manuscript Received June 15, 1990 abstract: A single-stranded DNA-dependent ATPase that cofractionates during the early stages of purification of a multiprotein DNA polymerase a complex fromHeLa cells has been purified to homogeneity. The ATPase is part of a 16S multienzyme DNA polymerase a complex that is fully active in SV40 DNA replication in vitro. The ATPasehydrolyzes ATP to ADP in a reaction that is completely dependent on the presence of DNA. DNA in single-stranded form is strongly preferred as a cofactor, and polydeoxynucleotides with adenine or thymidine residues are highly effective. Glycerol gradient sedimentation showed that the purified ATPasesedimented at an 52o, w of 7 S, and polyacrylamide gel electrophoresis under denaturing conditions reveals two polypeptides with relative molecular weights of 83 000 and 68 000. Both of these polypeptides have purine nucleotide binding sites as revealed by photoaffinity cross-linking ex-periments. ATP binds to the two subunits more efficiently than GTP, and CTP or UTP does not cross-link with the two polypeptides. DNA synthesis catalyzed by purified HeLa cell DNA polymerase a-primase is stimulated in the presence of ATPase and ATP at an optimum concentration of 2 mM. Analysis of the DNA product by gel electrophoresis indicates that with poly (dT) but not phage Ml3 DNA as template the ATPase overcomes a lag and decreases the length of nascent DNA chains synthesized by the DNA polymerase a-primase complex.