DNA-Segment-Facilitated Dissociation of Fis and NHP6A from DNA Detected via Single-Molecule Mechanical Response
DNA-Segment-Facilitated Dissociation of Fis and NHP6A from DNA Detected via Single-Molecule Mechanical Response
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DOI：
10.1016/j.jmb.2015.07.015
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发表时间：
2015-09-25
影响因子：
5.6
通讯作者：
Marko, John F.
中科院分区：
文献类型：
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作者：
Giuntoli, Rebecca D.;Linzer, Nora B.;Marko, John F.
DNA蛋白质复合物的解离速率通常被认为是该复合物的性质，而不依赖于溶液中的其他邻近分子。我们研究了大量的大肠杆菌类核蛋白Fis从DNA中解离的动力学，使用单分子力学测定。Fis从DNA中解离的速率强烈依赖于DNA的溶液浓度。Fis与DNA的解离速率（k（off））显示出对溶液DNA浓度的初始线性依赖性，其特征在于对于100 mM单价盐缓冲液，k（ex）的交换速率约为9 × 10（-4）（ng/μ l）（-1）s（-1），在0 DNA浓度下具有非常小的解离速率。当DNA浓度高于约20 ng/μ l时，解离速率在约8 x 10（-3）s（-1）的k（off，max）处饱和。这种交换反应主要取决于DNA浓度，而几乎不依赖于溶液中DNA分子的长度或结合亲和力，但这确实随着盐浓度的增加而增加。我们还显示了酵母HMGB蛋白NHP 6A的数据，显示了类似的DNA浓度依赖性解离效应，更快的速率表明NHP 6A相对于Fis的DNA结合通常较弱。我们的结果很好地描述了一个模型与中间部分解离状态的蛋白质是容易被捕获的第二个DNA片段，在“直接转移”的方式研究其他DNA结合蛋白的反应。这种类型的解离途径可能是重要的蛋白质DNA结合动力学在体内的DNA浓度很大。(C)2015爱思唯尔有限公司版权所有。
The rate of dissociation of a DNA protein complex is often considered to be a property of that complex, without dependence on other nearby molecules in solution. We study the kinetics of dissociation of the abundant Escherichia coli nucleoid protein Fis from DNA, using a single-molecule mechanics assay. The rate of Fis dissociation from DNA is strongly dependent on the solution concentration of DNA. The off-rate (k(off)) of Fis from DNA shows an initially linear dependence on solution DNA concentration, characterized by an exchange rate of k(ex) approximate to 9 x 10(-4) (ng/mu l)(-1) s(-1) for 100 mM univalent salt buffer, with a very small off-rate at zero DNA concentration. The off-rate saturates at approximately k(off,max) approximate to 8 x 10(-3) s(-1) for DNA concentrations above approximate to 20 ng/mu l. This exchange reaction depends mainly on DNA concentration with little dependence on the length of the DNA molecules in solution or on binding affinity, but this does increase with increasing salt concentration. We also show data for the yeast HMGB protein NHP6A showing a similar DNA-concentration-dependent dissociation effect, with faster rates suggesting generally weaker DNA binding by NHP6A relative to Fis. Our results are well described by a model with an intermediate partially dissociated state where the protein is susceptible to being captured by a second DNA segment, in the manner of "direct transfer" reactions studied for other DNA-binding proteins. This type of dissociation pathway may be important to protein DNA binding kinetics in vivo where DNA concentrations are large. (C) 2015 Elsevier Ltd. All rights reserved.