Olson M W, Wang Y, Elder R H, Kaguni L S
Department of Biochemistry, Michigan State University, East Lansing 48824, USA.
J Biol Chem. 1995 Dec 1;270(48):28932-7. doi: 10.1074/jbc.270.48.28932.
The subunit structure of mitochondrial DNA polymerase from Drosophila embryos has been examined by a combination of physical and immunological methods. A highly specific rabbit antiserum directed against the native enzyme was developed and found to recognize specifically its two subunits in immunoblot and immunoprecipitation analyses. That and the potent inhibition by the rabbit antiserum of the DNA polymerase and 3'-->5' exonuclease activities of the nearly homogeneous mitochondrial DNA polymerase provide strong evidence for the physical association of the 3'-->5' exonuclease with the two subunit enzyme. An immunoprecipitation analysis of crude enzyme fractions showed that the two subunits of Drosophila mitochondrial DNA polymerase are intact, and an in situ gel proteolysis analysis showed that they are structurally distinct. Template-primer DNA binding studies demonstrated formation of a stable and discrete enzyme-DNA complex in the absence of accessory proteins. Photochemical cross-linking of the complexes by UV light indicated that the alpha but not the beta subunit of mitochondrial DNA polymerase makes close contact with DNA, and limited digestion of the native enzyme with trypsin showed that an approximately 65-kDa proteolytic fragment of the alpha subunit retains the DNA binding function.
通过物理和免疫学方法相结合，对果蝇胚胎线粒体DNA聚合酶的亚基结构进行了研究。制备了一种针对天然酶的高度特异性兔抗血清，并发现其在免疫印迹和免疫沉淀分析中能特异性识别该酶的两个亚基。兔抗血清对几乎纯的线粒体DNA聚合酶的DNA聚合酶和3'→5'核酸外切酶活性具有强力抑制作用，这为3'→5'核酸外切酶与双亚基酶的物理关联提供了有力证据。对粗酶组分的免疫沉淀分析表明，果蝇线粒体DNA聚合酶的两个亚基是完整的，原位凝胶蛋白酶解分析表明它们在结构上是不同的。模板 - 引物DNA结合研究表明，在没有辅助蛋白的情况下形成了稳定且离散的酶 - DNA复合物。通过紫外线对复合物进行光化学交联表明，线粒体DNA聚合酶的α亚基而非β亚基与DNA紧密接触，用胰蛋白酶对天然酶进行有限消化表明，α亚基约65 kDa的蛋白水解片段保留了DNA结合功能。
