Chilkova Olga, Jonsson Bengt-Harald, Johansson Erik
Department of Medical Biochemistry and Biophysics, Umeå University, Sweden.
J Biol Chem. 2003 Apr 18;278(16):14082-6. doi: 10.1074/jbc.M211818200. Epub 2003 Feb 5.
DNA polymerase epsilon (Pol epsilon) from Saccharomyces cerevisiae consists of four subunits (Pol2, Dpb2, Dpb3, and Dpb4) and is essential for chromosomal DNA replication. Biochemical characterizations of Pol epsilon have been cumbersome due to protease sensitivity and the limited amounts of Pol epsilon in cells. We have developed a protocol for overexpression and purification of Pol epsilon from S. cerevisiae. The native four-subunit complex was purified to homogeneity by conventional chromatography. Pol epsilon was characterized biochemically by sedimentation velocity experiments and gel filtration experiments. The stoichiometry of the four subunits was estimated to be 1:1:1:1 from colloidal Coomassie-stained gels. Based on the sedimentation coefficient (11.9 S) and the Stokes radius (74.5 A), a molecular mass for Pol epsilon of 371 kDa was calculated, in good agreement with the calculated molecular mass of 379 kDa for a heterotetramer. Furthermore, analytical equilibrium ultracentrifugation experiments support the proposed heterotetrameric structure of Pol epsilon. Thus, both DNA polymerase delta and Pol epsilon are purified as monomeric complexes, in agreement with accumulating evidence that Pol delta and Pol epsilon are located on opposite strands of the eukaryotic replication fork.
酿酒酵母的DNA聚合酶ε（Pol ε）由四个亚基（Pol2、Dpb2、Dpb3和Dpb4）组成，对染色体DNA复制至关重要。由于蛋白酶敏感性以及细胞中Pol ε含量有限，对其进行生化特性分析一直很麻烦。我们开发了一种从酿酒酵母中过表达和纯化Pol ε的方案。通过常规色谱法将天然四亚基复合物纯化至同质。通过沉降速度实验和凝胶过滤实验对Pol ε进行生化特性分析。从考马斯亮蓝染色的凝胶中估计四个亚基的化学计量比为1:1:1:1。根据沉降系数（11.9 S）和斯托克斯半径（74.5 Å），计算出Pol ε的分子量为371 kDa，与异源四聚体计算出的379 kDa分子量非常吻合。此外，分析型平衡超速离心实验支持了所提出的Pol ε异源四聚体结构。因此，DNA聚合酶δ和Pol ε均作为单体复合物纯化，这与越来越多的证据一致，即Pol δ和Pol ε位于真核复制叉的相反链上。
