Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Methods. 2010 Aug;51(4):379-84. doi: 10.1016/j.ymeth.2010.02.015. Epub 2010 Feb 20.
More than 150 different point mutations in POLG, the gene encoding the human mitochondrial DNA polymerase gamma (pol gamma), cause a broad spectrum of childhood and adult onset diseases like Alpers syndrome, ataxia-neuropathy syndrome and progressive external ophthalmoplegia. These disease mutations can affect the pol gamma enzyme's properties in numerous ways, thus potentially influencing the severity of the disease. Hence, a detailed characterization of disease mutants will greatly assist researchers and clinicians to develop a clear understanding of the functional defects caused by these mutant enzymes. Experimental approaches for characterizing the wild-type (WT) and mutant pol gamma enzymes are extensively described in this manuscript. The methods start with construction and purification of the recombinant wild-type and mutant forms of pol gamma protein, followed by assays to determine its structural integrity and thermal stability. Next, the biochemical characterization of these enzymes is described in detail, which includes measuring the purified enzyme's catalytic activity, its steady-state kinetic parameters and DNA binding activity, and determining the physical and functional interaction of these pol gamma proteins with the p55 accessory subunit.
超过 150 种不同的 POLG 点突变，即编码人类线粒体 DNA 聚合酶γ（pol γ）的基因，导致广泛的儿童和成人发病疾病，如 Alpers 综合征、共济失调-感觉神经病和进行性眼外肌麻痹。这些疾病突变可以通过多种方式影响 pol γ 酶的特性，从而可能影响疾病的严重程度。因此，对疾病突变体进行详细的特征描述将极大地帮助研究人员和临床医生清楚地了解这些突变酶引起的功能缺陷。本手稿中详细描述了用于表征野生型（WT）和突变型 pol γ 酶的实验方法。这些方法从构建和纯化重组野生型和突变型 pol γ 蛋白开始，然后进行测定其结构完整性和热稳定性的实验。接下来，详细描述了这些酶的生化特征，包括测量纯化酶的催化活性、其稳态动力学参数和 DNA 结合活性，并确定这些 pol γ 蛋白与 p55 辅助亚基的物理和功能相互作用。
