Analyzing the forces binding a restriction endonuclease to DNA using a synthetic nanopore.
Analyzing the forces binding a restriction endonuclease to DNA using a synthetic nanopore.
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DOI：
10.1093/nar/gkp317
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发表时间：
2009-07
影响因子：
14.9
通讯作者：
Timp G
中科院分区：
文献类型：
--
作者：
Dorvel B;Sigalov G;Zhao Q;Comer J;Dimitrov V;Mirsaidov U;Aksimentiev A;Timp G
限制性内切酶在重组DNA技术中被广泛使用，因为它们稳定地结合到特定的靶序列上，并且在辅因子存在的情况下，以很高的速度在靶序列上特异地切割双螺旋DNA。利用合成的纳米孔和分子动力学(MD)，我们以原子分辨率分析了在没有镁离子辅助因子的情况下，典型的限制性内切酶EcoRI如何与DNA靶序列GAATTC结合。我们先前已经证明，与限制性内切酶结合的DNA通过纳米孔渗透有一个电压阈值，这与打破复合体所需的纳米牛子力有关。通过在DNA中引入突变，我们现在证明了这个阈值取决于识别序列，并与解离能成线性关系，与孔几何无关。为了预测碱基对突变对解离自由能的影响，MD被用来定性地对EcoRI-DNA复合体中键的稳定性进行排名。我们发现目标序列中的第二个碱基与蛋白质表现出最强的结合，其次是第三个和第一个碱基，甚至侧翼序列也影响结合，这证实了我们的实验。
Restriction endonucleases are used prevalently in recombinant DNA technology because they bind so stably to a specific target sequence and, in the presence of cofactors, cleave double-helical DNA specifically at a target sequence at a high rate. Using synthetic nanopores along with molecular dynamics (MD), we have analyzed with atomic resolution how a prototypical restriction endonuclease, EcoRI, binds to the DNA target sequence—GAATTC—in the absence of a Mg2+ ion cofactor. We have previously shown that there is a voltage threshold for permeation of DNA bound to restriction enzymes through a nanopore that is associated with a nanonewton force required to rupture the complex. By introducing mutations in the DNA, we now show that this threshold depends on the recognition sequence and scales linearly with the dissociation energy, independent of the pore geometry. To predict the effect of mutation in a base pair on the free energy of dissociation, MD is used to qualitatively rank the stability of bonds in the EcoRI–DNA complex. We find that the second base in the target sequence exhibits the strongest binding to the protein, followed by the third and first bases, with even the flanking sequence affecting the binding, corroborating our experiments.
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影响因子：
10.8
作者：
Fologea, D;Uplinger, J;Li, JL
通讯作者：
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影响因子：
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作者：
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DOI：
10.1073/pnas.0500796102
发表时间：
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影响因子：
11.1
作者：
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通讯作者：
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