Purification and Comparative Assay of the Human Mitochondrial Replicative DNA Helicase.
Purification and Comparative Assay of the Human Mitochondrial Replicative DNA Helicase.
复制标题
人线粒体复制 DNA 解旋酶的纯化和比较测定。
DOI：
10.1007/978-1-4939-3040-1_14
复制
发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Kaguni,LaurieS
中科院分区：
文献类型：
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作者：
Rosado-Ruiz,FernandoA;So,Minyoung;Kaguni,LaurieS
复制型线粒体DNA(MtDNA)解旋酶是线粒体基因组复制和维持所必需的。尽管它的特性已经取得了实质性的进展，但关于它的结构域的功能作用以及它与最小线粒体DNA复制体的其他成分的相互作用仍有许多需要了解。实现这一点的关键是以稳定、有活性的形式分离酶的能力。在这一章中，我们描述了一种改进的、简化的重组酶形式的纯化策略。我们还提供了评估其螺旋解旋活性和线粒体单链DNA结合蛋白(MtSSB)刺激作用的方法。最后，我们描述了一种浓度/缓冲液交换方法，我们已经使用这种方法来获得更好的酶稳定性和合适的生化和生物物理研究条件。
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.