Enzymological characterization of DNA polymerase alpha. Basic catalytic properties processivity, and gap utilization of the homogeneous enzyme from human KB cells.
Enzymological characterization of DNA polymerase alpha. Basic catalytic properties processivity, and gap utilization of the homogeneous enzyme from human KB cells.
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DNA聚合酶α的酶学表征。
DOI：
10.1016/s0021-9258(18)50528-7
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发表时间：
1979
期刊：
影响因子：
--
通讯作者：
D. Korn
中科院分区：
文献类型：
--
作者：
P. Fisher;T. S. Wang;D. Korn
本报告描述了我们从培养的人KB细胞中纯化的DNA聚合酶α的均匀制备的初步酶学表征的结果。尽管在需要Mg 2+的孵育中，该酶与含有短间隙（最佳活化）的双链DNA底物反应性最强，但该聚合酶具有以低速率复制起始核糖均聚物模板（A）-n，（dT）-200的内在能力。在Mn 2+的存在下。由于DNA聚合酶α在活跃增殖的脊椎动物细胞中占优势，很可能这种低水平的活性包含了可以在粗组织提取物中检测到的大部分核糖聚合物复制活性。可以从纯化的酶中排除污染或相关脱氧核糖核酸酶活性的存在，使其达到10（-4）至10（-7）的聚合酶活性水平。在最佳条件下，活化DNA的聚合机制是适度进行性的，每个聚合循环掺入11 +/-5个核苷酸。该聚合酶不能在切口或长度约20至30个核苷酸的短缺口处工作，并且它在最佳活化的DNA和已经用大肠杆菌核酸外切酶III缺口到不同程度的DNA分子上测量令人惊讶的不变的有效模板长度。在“附录”中，我们提出了一个放大的理论公式的Bambara等人（Bambara，R。一、Uyemura，D.，和Choi，T.（1978）J.Biol.Chem.253，413-423），其允许使用具有显著相关的3'导致5'-外切核酸酶活性的DNA聚合酶来精确测量平均模板长度（缺口大小）和滴定缺口双链体DNA底物中的可用3 '-羟基引物末端。
This report describes the results of our initial enzymological characterization of a homogeneous preparation of DNA polymerase alpha that we have purified from cultured human KB cells. Although the enzyme is most reactive with duplex DNA substrates that contain short gaps (optimally activated) in incubations that require Mg2+, the polymerase possesses the intrinsic capacity to copy the initiated ribohomopolymer template, (A)-n, (dT)-200, at low rates in the presence of Mn2+. Because of the preponderance of DNA polymerase alpha in actively multiplying vertebrate cells, it is probable that this low level of activity comprises the majority of the ribopolymer copying activity that can be detected in crude tissue extracts. The presence of contaminating or associated deoxyribonuclease activities can be excluded from the purified enzyme to levels of 10(-4) to 10(-7) of the polymerase activity. The mechanism of polymerization on activated DNA under optimum conditions is moderately processive, with 11 +/- 5 nucleotides incorporated per polymerization cycle. The polymerase is unable to work at nicks or at short gaps of approximately 20 to 30 nucleotides in length, and it measures a surprisingly invariant effective template length on optimally activated DNA and on DNA molecules that have been gapped to varying extents with Escherichia coli exonuclease III. In the “Appendix” we present an amplification of the theoretical formulation of Bambara et al. (Bambara, R. A., Uyemura, D., and Choi, T. (1978) J. Biol. Chem. 253, 413–423) that permits the use of DNA polymerases with significant associated 3' leads to 5'-exonuclease activities for the accurate measurement of average template lengths (gap sizes) and titration of usable 3'-hydroxyl primer termini in gapped, duplex DNA substrates.