人线粒体复制性DNA解旋酶的纯化及比较分析
Purification and Comparative Assay of the Human Mitochondrial Replicative DNA Helicase.
作者信息
Rosado-Ruiz Fernando A, So Minyoung, Kaguni Laurie S
机构信息
Department of Biochemistry and Molecular Biology, Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, MI, 48824, USA.
Institute of Biosciences of Medical Technology, University of Tempere, Tempere, 33014, Finland.
出版信息
Methods Mol Biol. 2016;1351:185-98. doi: 10.1007/978-1-4939-3040-1_14.
The replicative mitochondrial DNA (mtDNA) helicase is essential for mtDNA replication and maintenance of the mitochondrial genome. Despite substantial advances that have been made in its characterization, there is still much to be understood about the functional roles of its domains and its interactions with the other components of the minimal mitochondrial DNA replisome. Critical to achieving this is the ability to isolate the enzyme in a stable, active form. In this chapter we describe a modified, streamlined purification strategy for recombinant forms of the enzyme. We also present assays to assess its helix unwinding activity and the stimulatory effects of the mitochondrial single-stranded DNA-binding protein (mtSSB). Finally, we describe a concentration/buffer exchange method that we have employed to achieve greater enzyme stability and appropriate conditions for biochemical and biophysical studies.
复制性线粒体DNA（mtDNA）解旋酶对于mtDNA复制和线粒体基因组的维持至关重要。尽管在其特性表征方面已取得重大进展，但关于其结构域的功能作用以及与最小线粒体DNA复制体其他组分的相互作用，仍有许多有待了解之处。实现这一目标的关键在于能够以稳定、有活性的形式分离该酶。在本章中，我们描述了一种针对该酶重组形式的改良、简化纯化策略。我们还介绍了评估其解螺旋活性以及线粒体单链DNA结合蛋白（mtSSB）刺激作用的检测方法。最后，我们描述了一种用于实现更高酶稳定性以及适用于生化和生物物理研究条件的浓缩/缓冲液交换方法。
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