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Immunoaffinity purification of the 180-kDa and 86-kDa subunits and in vitro reconstitution of the Saccharomyces cerevisiae DNA primase-DNA polymerase protein complex.

机译：180 kDa和86 kDa亚基的免疫亲和纯化和酿酒酵母DNA primase-DNA聚合酶蛋白复合物的体外重组。

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The DNA primase-DNA polymerase protein complex is required for nuclear chromosomal DNA replication in the yeast S. cerevisiae. This complex is composed of four subunits, 180-, 86-, 58-, and 49-kDa. The DNA primase heterodimer (58-kDa and 49-kDa) and associated enzymatic activity have been previously studied. The catalytic DNA polymerase 180-kDa subunit and the 86-kDa subunit polypeptides have been purified using immunoaffinity chromatography to apparent homogeneity from separate overproducing yeast strains using monoclonal antibodies specifically recognizing each subunit. A four subunit reconstituted complex was detected when the 180-kDa, 86-kDa, and DNA primase heterodimer were coincubated in vitro. The reconstituted four subunit complex behaved as a single species, exhibiting a Stokes radius of 80A and a sedimentation coefficient of 8.9S. I infer from the calculated molecular weight of 312,000 that the stoichiometry is one of each subunit per complex.;While the 86-kDa subunit readily forms a physical complex with the 180-kDa DNA polymerase catalytic subunit, I have not detected a complex containing only the 86-kDa and the DNA primase heterodimer. When the 180-kDa and DNA primase subunits were recombined in the absence of the 86-kDa subunit, a physical complex formed. Therefore, the 86-kDa subunit was not an obligatory "linker" subunit for complex formation. However, 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 associated with DNA polymerase activity.;The isolated 180-kDa polypeptide was sufficient to catalyze all the DNA synthesis which had been previously observed in the DNA primase-DNA polymerase protein complex. The apparent DNA polymerase processivity was not affected by the presence of the 86-kDa subunit, but was reduced by increased Mg

机译：DNA primase-DNA聚合酶蛋白复合物是酵母酵母中核染色体DNA复制所必需的。该复合物由180 kDa，86 kDa，58 kDa和49 kDa四个亚基组成。 DNA primase异源二聚体（58-kDa和49-kDa）和相关的酶活性已在先前进行了研究。已使用免疫亲和色谱法纯化了催化性DNA聚合酶180-kDa亚基和86-kDa亚基多肽，以使用特异识别每个亚基的单克隆抗体从单独的过量生产酵母菌株中获得明显的同质性。当180 kDa，86 kDa和DNA primase异源二聚体在体外共孵育时，检测到四个亚基重组复合物。重构的四个亚基复合物表现为单一物种，其斯托克斯半径为80A，沉降系数为8.9S。我从计算出的分子量312,000推论出化学计量是每个复合物中每个亚基之一;虽然86-kDa亚基很容易与180-kDa DNA聚合酶催化亚基形成物理复合物，但我没有检测到仅包含一个复合物的复合物。 86 kDa和DNA primase异二聚体。当在没有86-kDa亚基的情况下重组180-kDa和DNA primase酶亚基时，形成了物理复合物。因此，86 kDa亚基不是复合物形成的强制性“接头”亚基。然而，86-kDa亚基增加了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。;分离的180-kDa多肽足以催化以前在DNA primase-DNA polymerase蛋白复合物中观察到的所有DNA合成。表观的DNA聚合酶的合成能力不受86-kDa亚基的影响，但会因Mg增加而降低

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作者
Brooke, Richard Glenn.;
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Northwestern University.;

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授予单位 Northwestern University.;
学科 Molecular biology.;Genetics.
学位 Ph.D.
年度 1990
页码 134 p.
总页数 134
原文格式 PDF
正文语种 eng
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1. The small subunits of human and mouse DNA polymerase epsilon are homologous to the second largest subunit of the yeast Saccharomyces cerevisiae DNA polymerase epsilon. [J] . Jokela M, Makiniemi M, Lehtonen S, Nucleic Acids Research . 1998,第3期

机译：人和小鼠DNA聚合酶epsilon的小亚基与酵母酿酒酵母DNA聚合酶epsilon的第二大亚基同源。
2. The small subunits of human and mouse DNA polymerase ε are homologous to the second largest subunit of the yeast Saccharomyces cerevisiae DNA polymerase ε [J] . Claude Szpirer, Juhani E. Syv?oja, Maarit Jokela, Nucleic acids research . 1998,第3期

机译：人和小鼠DNA聚合酶ε的小亚基与酵母酿酒酵母DNA聚合酶ε的第二大亚基同源
3. The Saccharomyces cerevisiae DNA polymerase alpha catalytic subunit interacts with Cdc68/Spt16 and with Pob3, a protein similar to an HMG1-like protein. [J] . J Wittmeyer, T Formosa Molecular and Cellular Biology . 1997,第7期

机译：酿酒酵母DNA聚合酶α催化亚基与Cdc68 / Spt16和与类似于HMG1蛋白的蛋白Pob3相互作用。
4. Towards an understanding of protein-protein interaction network hierarchies. Analysis of DnaN (β)-binding peptide motifs in members of protein families interacting with the eubacterial processivity clamp, the β subunit of DNA Polymerase III [C] . Brian P. Dalrymple, Gene Wijffels, Kritaya Kongsuwan, Asia-Pacific Bioinformatics Conference(APBC 2003); 200302; Adelaide(AU) . 2003

机译：对蛋白质-蛋白质相互作用网络层次结构的理解。蛋白质家族成员与DNA聚合酶III的优生过程性钳位相互作用的DnaN（β）结合肽基序分析
5. The redesign of a type 2 into a type 1 copper protein: Construction, expression, purification and characterization of metal-binding site mutant proteins of copper, zinc superoxide dismutase from Saccharomyces cerevisiae [D] . Lu, Yi. 1992

机译：将2型转变为1型铜蛋白：啤酒酵母中铜，锌超氧化物歧化酶的金属结合位点突变蛋白的构建，表达，纯化和表征
6. The small subunits of human and mouse DNA polymerase epsilon are homologous to the second largest subunit of the yeast Saccharomyces cerevisiae DNA polymerase epsilon. [O] . M Jokela, M Mäkiniemi, S Lehtonen, 1998

机译：人和小鼠DNA聚合酶ε的小亚基与酵母酿酒酵母DNA聚合酶ε的第二大亚基同源。
7. In vivo reconstitution of Saccharomyces cerevisiae DNA polymerase epsilon in insect cells - Purification and characterization [O] . Dua Rajiv, Levy Daniel L., Li Caroline M., 2002

机译：昆虫细胞中啤酒酵母DNA聚合酶ε的体内重组-纯化和表征

Immunoaffinity purification of the 180-kDa and 86-kDa subunits and in vitro reconstitution of the Saccharomyces cerevisiae DNA primase-DNA polymerase protein complex.

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