Vishwanatha J K, Baril E F
Department of Biochemistry, University of Nebraska Medical Center, Omaha 68198.
Biochemistry. 1990 Sep 18;29(37):8753-9. doi: 10.1021/bi00489a036.
A single-stranded DNA-dependent ATPase that cofractionates during the early stages of purification of a multiprotein DNA polymerase alpha complex from HeLa cells has been purified to homogeneity. The ATPase is part of a 16S multienzyme DNA polymerase alpha complex that is fully active in SV40 DNA replication in vitro. The ATPase hydrolyzes 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 ATPase sedimented at an s20,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 experiments. 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 alpha-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 M13 DNA as template the ATPase overcomes a lag and decreases the length of nascent DNA chains synthesized by the DNA polymerase alpha-primase complex.
一种单链DNA依赖性ATP酶已被纯化至同质，该酶在从HeLa细胞中纯化多蛋白DNA聚合酶α复合物的早期阶段与其他成分共同分级分离。该ATP酶是16S多酶DNA聚合酶α复合物的一部分，在体外SV40 DNA复制中具有完全活性。该ATP酶在完全依赖DNA存在的反应中将ATP水解为ADP。单链形式的DNA作为辅因子是强烈优选的，具有腺嘌呤或胸腺嘧啶残基的多脱氧核苷酸非常有效。甘油梯度沉降显示纯化的ATP酶在s20,w为7 S处沉降，变性条件下的聚丙烯酰胺凝胶电泳显示出两条相对分子量分别为83,000和68,000的多肽。光亲和交联实验表明这两条多肽都具有嘌呤核苷酸结合位点。ATP比GTP更有效地结合到这两个亚基上，而CTP或UTP不会与这两条多肽交联。在ATP酶和2 mM最佳浓度的ATP存在下，纯化的HeLa细胞DNA聚合酶α-引发酶催化的DNA合成受到刺激。通过凝胶电泳对DNA产物的分析表明，以聚（dT）而不是噬菌体M13 DNA为模板时，ATP酶克服了滞后现象并缩短了由DNA聚合酶α-引发酶复合物合成的新生DNA链的长度。
