人线粒体单链DNA结合蛋白的纯化及比较分析
Purification and Comparative Assay of Human Mitochondrial Single-Stranded DNA-Binding Protein.
作者信息
Ciesielski Grzegorz L, Rosado-Ruiz Fernando A, Kaguni Laurie S
机构信息
Institute of Biosciences and Medical Technology, University of Tampere, Tampere, 33014, Finland.
Department of Biochemistry and Molecular Biology, Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, MI, 48824-1319, USA.
出版信息
Methods Mol Biol. 2016;1351:211-22. doi: 10.1007/978-1-4939-3040-1_16.
Abstract
The mitochondrial single-stranded DNA-binding protein (mtSSB) coordinates the function of replisome components at the mitochondrial replication fork. In recent years, it has been demonstrated that mtSSB stimulates the activities of DNA polymerase γ (Pol γ) and mitochondrial DNA (mtDNA) helicase in a concentration-dependent manner. Here we present a new approach to purify the human mtSSB and our standard assays to evaluate its biochemical properties, including a Gel Mobility Shift Assay (GMSA) to assess single-stranded DNA (ssDNA) binding activity, and an assay to assess SSB stimulation of Pol γ activity.
摘要
线粒体单链DNA结合蛋白（mtSSB）在线粒体复制叉处协调复制体组分的功能。近年来，已证明mtSSB以浓度依赖的方式刺激DNA聚合酶γ（Pol γ）和线粒体DNA（mtDNA）解旋酶的活性。在此，我们提出一种纯化人mtSSB的新方法以及评估其生化特性的标准检测方法，包括用于评估单链DNA（ssDNA）结合活性的凝胶迁移率变动分析（GMSA），以及用于评估SSB对Pol γ活性刺激作用的检测方法。
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