Biswas E E, Chen P H, Gray W, Li Y H, Ray S, Biswas S B
Department of Pediatrics, University of Maryland School of Medicine, Baltimore 21201.
Biochemistry. 1993 Mar 30;32(12):3013-9. doi: 10.1021/bi00063a012.
We have purified a multimeric form of yeast DNA polymerase alpha with DNA polymerase, primase, 5'-->3' exonuclease, and single-stranded (ss) DNA-dependent ATPase activities to near-homogeneity. The molecular mass of complex was 650 kDa with subunits ranging in sizes from 30 to 180 kDa. The alpha-subunit of the complex could be detected by DNA polymerase alpha antibody. No cross-reactivity of polypeptides within the complex was observed with antibodies directed against polymerase delta or epsilon. The multimeric polymerase alpha could be selectively inhibited by p-n-butylphenyl-dGTP (I50 of approximately 0.2 microM), p-n-butylanilino-dATP (I50 of 1.3 microM), and aphidicolin (I50 of 2.5 micrograms/mL). The complex synthesized RNA primers on various ssDNA templates and rapidly elongated these primers into nascent DNA fragments in the presence of required deoxynucleotides. It has a strong 5'-->3' exonuclease activity. In addition, the complex hydrolyzed both ATP and dATP in a ssDNA-dependent manner. Thus, the multiprotein complex of DNA polymerase alpha had multiple activities (primase, polymerase, and ATPase) which could act concertedly to synthesize primers and elongate the primers to nascent DNA fragments in the lagging strand of the fork.
我们已经将一种具有DNA聚合酶、引发酶、5'→3'核酸外切酶和单链（ss）DNA依赖性ATP酶活性的多聚体形式的酵母DNA聚合酶α纯化至接近均一。该复合物的分子量为650 kDa，亚基大小范围从30 kDa到180 kDa。该复合物的α亚基可通过DNA聚合酶α抗体检测到。用针对聚合酶δ或ε的抗体未观察到复合物内多肽的交叉反应性。多聚体DNA聚合酶α可被对正丁基苯基-dGTP（I50约为0.2 microM）、对正丁基苯胺基-dATP（I50为1.3 microM）和阿非迪霉素（I50为2.5微克/毫升）选择性抑制。该复合物在各种ssDNA模板上合成RNA引物，并在存在所需脱氧核苷酸的情况下将这些引物迅速延伸为新生DNA片段。它具有很强的5'→3'核酸外切酶活性。此外，该复合物以ssDNA依赖性方式水解ATP和dATP。因此，DNA聚合酶α的多蛋白复合物具有多种活性（引发酶、聚合酶和ATP酶），这些活性可协同作用以合成引物并将引物延伸至叉状结构后随链中的新生DNA片段。
