Brooke R G, Dumas L B
Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208.
J Biol Chem. 1991 Jun 5;266(16):10093-8.
The immunoaffinity-purified subunits of the yeast DNA primase-DNA polymerase protein complex and subunit-specific monoclonal antibodies were used to explore the structural relationships of the subunits in the complex. The reconstituted four-subunit complex (180-, 86-, 58-, and 49-kDa polypeptides) behaved as a single species, exhibiting a Stokes radius of 80 A and a sedimentation coefficient of 8.9 S. The calculated molecular weight of the reconstituted complex is 312,000. We infer that the stoichiometry of the complex is one of each subunit per complex. The complex has a prolate ellipsoid shape with an axial ratio of approximately 16. When the 180-kDa and DNA primase subunits were recombined in the absence of the 86-kDa subunit, a physical complex formed, as judged by immunoprecipitation of DNA primase activity and polypeptides with an anti-180-kDa monoclonal antibody. While the 86-kDa subunit readily forms a physical complex with the 180-kDa DNA polymerase catalytic subunit, we have not detected a complex containing 86-kDa and the DNA primase subcomplex (49- and 58-kDa subunits). The 86-kDa subunit was not required for DNA primase-DNA polymerase complex formation; the 180-kDa subunit and DNA primase heterodimer directly interact. However, the presence of the 86-kDa subunit increased the rate at which the DNA primase and 180-kDa polypeptides formed a complex and increased the total fraction of DNA primase activity that was associated with DNA polymerase activity. The observations demonstrate that the DNA primase p49.p58 heterodimer and the DNA polymerase p86.p180 heterodimer interact via the 180-kDa subunit. The four-subunit reconstituted complex was sufficient to catalyze the DNA chain extension coupled to RNA primer synthesis on a single-stranded DNA template, as previously observed in the conventionally purified complex isolated from wild type cells.
利用免疫亲和纯化的酵母DNA引发酶 - DNA聚合酶蛋白复合物的亚基和亚基特异性单克隆抗体，来探究该复合物中亚基的结构关系。重组的四亚基复合物（180 kDa、86 kDa、58 kDa和49 kDa的多肽）表现为单一物种，斯托克斯半径为80 Å，沉降系数为8.9 S。重组复合物的计算分子量为312,000。我们推断该复合物的化学计量比是每个复合物中各有一个亚基。该复合物呈长椭圆形，轴比约为16。当180 kDa亚基和DNA引发酶亚基在没有86 kDa亚基的情况下重组时，形成了一个物理复合物，这是通过用抗180 kDa单克隆抗体免疫沉淀DNA引发酶活性和多肽来判断的。虽然86 kDa亚基很容易与180 kDa的DNA聚合酶催化亚基形成物理复合物，但我们尚未检测到包含86 kDa亚基和DNA引发酶亚复合物（49 kDa和58 kDa亚基）的复合物。DNA引发酶 - DNA聚合酶复合物的形成不需要86 kDa亚基；180 kDa亚基和DNA引发酶异二聚体直接相互作用。然而，86 kDa亚基的存在增加了DNA引发酶和180 kDa多肽形成复合物的速率，并增加了与DNA聚合酶活性相关的DNA引发酶活性的总比例。这些观察结果表明，DNA引发酶p49.p58异二聚体和DNA聚合酶p86.p180异二聚体通过180 kDa亚基相互作用。如先前在从野生型细胞中分离得到的传统纯化复合物中所观察到的那样，重组的四亚基复合物足以催化与单链DNA模板上的RNA引物合成偶联的DNA链延伸。
