利用荧光各向异性测定腺相关病毒Rep DNA结合情况
Determination of Adeno-associated Virus Rep DNA Binding Using Fluorescence Anisotropy.
作者信息
Zarate-Perez Francisco, Santosh Vishaka, Bardelli Martino, Agundez Leticia, Linden R Michael, Henckaerts Els, Escalante Carlos R
机构信息
Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond VA, USA.
Department of Infectious Diseases, King's College London, London, United Kingdom.
出版信息
Bio Protoc. 2017 Mar 20;7(6). doi: 10.21769/BioProtoc.2194.
Abstract
Quantitative measurement of proteins binding to DNA is a requisite to fully characterize the structural determinants of complex formation necessary to understand the DNA transactions that regulate cellular processes. Here we describe a detailed protocol to measure binding affinity of the adeno-associated virus (AAV) Rep68 protein for the integration site using fluorescent anisotropy. This protocol can be used to measure the binding constants of any DNA binding protein provided the substrate DNA is fluorescently labeled.
摘要
对与DNA结合的蛋白质进行定量测量，是全面表征复合物形成的结构决定因素的必要条件，而理解调节细胞过程的DNA相互作用需要这些结构决定因素。在此，我们描述了一种详细的实验方案，用于使用荧光各向异性测量腺相关病毒（AAV）Rep68蛋白与整合位点的结合亲和力。只要底物DNA用荧光标记，该实验方案可用于测量任何DNA结合蛋白的结合常数。
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本文引用的文献
1
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J Virol. 2016 Jul 11;90(15):6612-6624. doi: 10.1128/JVI.00356-16. Print 2016 Aug 1.
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Structural Insights into the Assembly of the Adeno-associated Virus Type 2 Rep68 Protein on the Integration Site AAVS1.2型腺相关病毒Rep68蛋白在整合位点AAVS1上组装的结构见解
J Biol Chem. 2015 Nov 13;290(46):27487-99. doi: 10.1074/jbc.M115.669960. Epub 2015 Sep 14.
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DNA structure modulates the oligomerization properties of the AAV initiator protein Rep68.DNA结构调节腺相关病毒起始蛋白Rep68的寡聚化特性。
PLoS Pathog. 2009 Jul;5(7):e1000513. doi: 10.1371/journal.ppat.1000513. Epub 2009 Jul 10.
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Residues within the B' motif are critical for DNA binding by the superfamily 3 helicase Rep40 of adeno-associated virus type 2.B'基序内的残基对于2型腺相关病毒超家族3解旋酶Rep40与DNA的结合至关重要。
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The nuclease domain of adeno-associated virus rep coordinates replication initiation using two distinct DNA recognition interfaces.腺相关病毒rep的核酸酶结构域利用两个不同的DNA识别界面来协调复制起始。
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A maltose-binding protein/adeno-associated virus Rep68 fusion protein has DNA-RNA helicase and ATPase activities.一种麦芽糖结合蛋白/腺相关病毒Rep68融合蛋白具有DNA-RNA解旋酶和ATP酶活性。
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The AAV origin binding protein Rep68 is an ATP-dependent site-specific endonuclease with DNA helicase activity.腺相关病毒（AAV）的起始结合蛋白Rep68是一种具有DNA解旋酶活性的ATP依赖性位点特异性核酸内切酶。
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