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首页> 外文期刊>The FEBS journal >Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV
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Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV

机译：调节限制性核酸内切酶EcoRV的DNA结合特异性的溶液参数

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摘要

The DNA binding stringency of restriction endonucleases is crucial for their proper function. The X-ray structures of the specific and non-cognate complexes of the restriction nuclease EcoRV are considerably different suggesting significant differences in the hydration and binding free energies. Nonetheless, the majority of studies performed at pH 7.5, optimal for enzymatic activity, have found a < 10-fold difference between EcoRV binding constants to the specific and nonspecific sequences in the absence of divalent ions. We used a recently developed self-cleavage assay to measure EcoRV-DNA competitive binding and to evaluate the influence of water activity, pH and salt concentration on the binding stringency of the enzyme in the absence of divalent ions. We find the enzyme can readily distinguish specific and nonspecific sequences. The relative specific-nonspecific binding constant increases strongly with increasing neutral solute concentration and with decreasing pH. The difference in number of associated waters between specific and nonspecific DNA-EcoRV complexes is consistent with the differences in the crystal structures. Despite the large pH dependence of the sequence specificity, the osmotic pressure dependence indicates little change in structure with pH. The large osmotic pressure dependence means that measurement of protein-DNA specificity in dilute solution cannot be directly applied to binding in the crowded environment of the cell. In addition to divalent ions, water activity and pH are key parameters that strongly modulate binding specificity of EcoRV.
机译：限制性核酸内切酶的DNA结合严格性对于它们的正常功能至关重要。限制性核酸酶EcoRV的特异性和非同源复合物的X射线结构显着不同，表明水合和结合自由能存在显着差异。但是，在最适合酶促活性的pH值为7.5的条件下进行的大多数研究发现，在不存在二价离子的情况下，EcoRV与特定序列和非特定序列的结合常数之间的差异小于10倍。我们使用了最近开发的自切割测定法来测量EcoRV-DNA竞争结合并评估在没有二价离子的情况下水活度，pH和盐浓度对酶结合严格性的影响。我们发现该酶可以轻松地区分特定序列和非特定序列。相对特异性-非特异性结合常数随中性溶质浓度的增加和pH的降低而强烈增加。特异性和非特异性DNA-EcoRV复合物之间缔合水的数量差异与晶体结构的差异一致。尽管序列特异性对pH的依赖性很大，但渗透压依赖性表明随pH的结构变化很小。较大的渗透压依赖性意味着稀溶液中蛋白质-DNA特异性的测量不能直接应用于在拥挤的细胞环境中的结合。除二价离子外，水活度和pH值是强烈调节EcoRV结合特异性的关键参数。

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