Characterization of DNA Primase Complex Isolated from the Archaeon, Thermococcus kodakaraensis
Characterization of DNA Primase Complex Isolated from the Archaeon, Thermococcus kodakaraensis
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DOI：
10.1074/jbc.m111.338145
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发表时间：
2012-05-11
影响因子：
4.8
通讯作者：
Hurwitz, Jerard
中科院分区：
文献类型：
--
作者：
Galal, Wiebke Chemnitz;Pan, Miao;Hurwitz, Jerard
在大多数生物体中，DNA复制由DNA引物酶启动，DNA引物酶合成被DNA聚合酶延长的引物。在这项研究中，我们描述了来自Kodakaraensis古细菌热球菌的DNA引发酶复合物及其亚基的分离和生化特征。霸王Kodakaraensis DNA引发酶复合物是含有化学计量水平的p41和p46亚基的异二聚体。复合物的催化活性存在于p41亚基内。我们发现，该复合物支持DNA和RNA的合成，而p41亚基单独边际产生RNA和合成的DNA链比那些形成的复杂。我们报告说，T。Kodakaraensis引发酶复合物优先与dNTP而不是核糖核苷三磷酸相互作用，并从头启动RNA和DNA链。后者的研究结果表明，古细菌引发酶复合物，在真核生物的同源物相反，可以启动DNA链合成的情况下，核糖核苷三磷酸。由古菌复合体形成的DNA引物可以被T. kodakaraensis DNA聚合酶（Pol）B，而由p41催化亚基单独形成的DNA引物则不是。用p46亚基补充含有p41亚基的反应导致PolB催化的DNA合成。我们还建立了一个滚环反应，使用引物200核苷酸的环作为底物。在T.产生Kodakaraensis微型染色体维持（MCM）3 '-> 35' DNA解旋酶、PolB、复制因子C和增殖细胞核抗原、长前导链（>10 kb）。用DNA引发酶复合物补充这种反应也支持滞后链的形成。
In most organisms, DNA replication is initiated by DNA primases, which synthesize primers that are elongated by DNA polymerases. In this study, we describe the isolation and biochemical characterization of the DNA primase complex and its subunits from the archaeon Thermococcus kodakaraensis. The T. kodakaraensis DNA primase complex is a heterodimer containing stoichiometric levels of the p41 and p46 subunits. The catalytic activity of the complex resides within the p41 subunit. We show that the complex supports both DNA and RNA synthesis, whereas the p41 subunit alone marginally produces RNA and synthesizes DNA chains that are longer than those formed by the complex. We report that the T. kodakaraensis primase complex preferentially interacts with dNTP rather than ribonucleoside triphosphates and initiates RNA as well as DNA chains de novo. The latter findings indicate that the archaeal primase complex, in contrast to the eukaryote homolog, can initiate DNA chain synthesis in the absence of ribonucleoside triphosphates. DNA primers formed by the archaeal complex can be elongated extensively by the T. kodakaraensis DNA polymerase (Pol) B, whereas DNA primers formed by the p41 catalytic subunit alone were not. Supplementation of reactions containing the p41 subunit with the p46 subunit leads to PolB-catalyzed DNA synthesis. We also established a rolling circle reaction using a primed 200-nucleotide circle as the substrate. In the presence of the T. kodakaraensis minichromosome maintenance (MCM) 3'-> 35' DNA helicase, PolB, replication factor C, and proliferating cell nuclear antigen, long leading strands (>10 kb) are produced. Supplementation of such reactions with the DNA primase complex supported lagging strand formation as well.