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首页> 外文期刊>Biochemistry >Kinetic characterization of linear diffusion of the restriction endonuclease EcoRV on DNA
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Kinetic characterization of linear diffusion of the restriction endonuclease EcoRV on DNA

机译：限制性核酸内切酶EcoRV在DNA上线性扩散的动力学特征

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摘要

We have examined the kinetic parameters of linear diffusion of EcoRV on DNA. The data were analyzed by Monte Carlo simulations in which the efficiency of recognition of EcoRV sites during linear diffusion, the efficiency of Linear diffusion, and, the behavior of enzymes at the ends of linear DNA is explicitly treated, The analysis of the dependence of Linear diffusion on the concentrations of NaCl and MgCl2 shows that linear diffusion is maximal at 50 mM NaCl under all concentrations of MgCl2 tested and increases with increasing concentrations of Mg2+ up to 10 mM. the highest concentration used in the test. Under these conditions, EcoRV scans 2 x 10(6) bp during one binding event with a velocity of about 1.7 x 10(6) bp s(-1). The enzyme tends to overlook cleavage sites at 1 mM but not at 10 mM. MgCl2. This result confirms the thermodynamic finding that EcoRV does not bind very specifically to DNA in the absence of Mg2+. It demonstrates that there is a Mg2+-dependent continuous transition between a nonspecific and a specific binding mode of EcoRV to DNA, By comparing cleavage rates of linear DNA whose ends are free or blocked, we have shown that EcoRV has a very low probability to fall off at the ends of linear DNA. The enzyme rather is "reflected" and continues linear diffusion. EcoRV does not cleave oligonucleotides containing two EcoRV sites processively. Consequently, dissociation of the enzyme from the cleavage products is not preceded by a transfer to nonspecific DNA, and linear diffusion is not involved in product dissociation in EcoRV. [References: 61]
机译：我们已经检查了EcoRV在DNA上线性扩散的动力学参数。通过蒙特卡洛模拟分析数据，其中线性扩散过程中识别EcoRV位点的效率，线性扩散效率以及线性DNA末端酶的行为均得到明确处理。 NaCl和MgCl2浓度的扩散表明，在所有测试的MgCl2浓度下，线性扩散在50 mM NaCl时最大，并随Mg2 +浓度的增加而增加，直至10 mM。测试中使用的最高浓度。在这些条件下，EcoRV在一个结合事件中以大约1.7 x 10（6）bps（-1）的速度扫描2 x 10（6）bp。该酶倾向于忽略1 mM处的切割位点，而不会忽略10 mM处的切割位点。氯化镁该结果证实了热力学发现，即在缺少Mg2 +的情况下，EcoRV不能非常特异性地与DNA结合。它表明在EcoRV与DNA的非特异性和特异性结合模式之间存在Mg2 +依赖性连续过渡，通过比较末端为游离或受阻的线性DNA的裂解率，我们证明EcoRV掉落的可能性非常低。在线性DNA的末端。酶反而被“反射”并继续线性扩散。 EcoRV不会连续切割包含两个EcoRV位点的寡核苷酸。因此，酶从裂解产物上解离之前不会转移至非特异性DNA，并且EcoRV中产物解离中不涉及线性扩散。 [参考：61]

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