BamHI限制性内切核酸酶切割超螺旋DNA的时间分辨动力学。

The time-resolved kinetics of superhelical DNA cleavage by BamHI restriction endonuclease.

作者信息

Hensley P, Nardone G, Chirikjian J G, Wastney M E

机构信息

Macromolecular Sciences Department, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406-0939.

出版信息

J Biol Chem. 1990 Sep 5;265(25):15300-7.

PMID:2203774

Abstract

The kinetics of the cleavage of superhelical plasmid DNA (pBR322) by the restriction endonuclease, BamHI, have been analyzed in terms a compartmental model consistent with the chemistry first proposed by Rubin and Modrich (Rubin, R. A., and Modrich, P. (1978) Nucleic Acids Res. 5, 2991-2997) for analysis of the kinetics of the restriction endonuclease, EcoRI. The model was defined in terms of two compartments representing DNA substrate (bound and free), two compartments representing nicked intermediate (bound and free), one compartment representing linear product, and one compartment for free enzyme. A simultaneous analysis of concentration changes over time of the three DNA forms (superhelical, nicked, and linear) at six different enzyme concentrations was undertaken employing this compartmental model using SAAM (Simulation Analysis And Modeling) software. Results showed that rate constants characterizing the association of enzyme with superhelical DNA (6.0 x 10(5) M-1 s-1) and nicked DNA (2.8 x 10(5) M-1 s-1) were similar in magnitude and rate constants characterizing cleavage of the first (1.2 x 10(-2) s-1) and second phosphodiester bonds (3.1 x 10(-2) s-1) were also similar. The analysis yields a kinetically determined equilibrium constant of 12.9 nM for the dissociation of nicked intermediate from the enzyme. The rate constant describing the release of the nicked intermediate from the enzyme has a value of 3.7 x 10(-3) s-1. By comparing the value of this release rate constant to the value of the constant describing the second cleavage event, it can be determined that only 10% of the nicked intermediate bound to the enzyme is released as free nicked DNA and that 90% of the nicked intermediate is processed to the linear form without being released. Hence, most of the DNA is cleaved as the result of a single enzyme-DNA recognition event. No steady state assumptions were made in the analysis. The approach was to directly solve the differential equations which described the kinetic processes using an interactive method. This study demonstrates the usefulness of this approach for the analysis of kinetics of protein-DNA interactions for the restriction endonucleases.

摘要

利用一种区室模型分析了限制性内切核酸酶BamHI切割超螺旋质粒DNA（pBR322）的动力学，该模型与Rubin和Modrich最初提出的用于分析限制性内切核酸酶EcoRI动力学的化学模型相一致（Rubin, R. A., and Modrich, P. (1978) Nucleic Acids Res. 5, 2991 - 2997）。该模型定义了两个代表DNA底物（结合态和游离态）的区室、两个代表带切口中间体（结合态和游离态）的区室、一个代表线性产物的区室以及一个代表游离酶的区室。使用SAAM（模拟分析与建模）软件，采用这个区室模型对六种不同酶浓度下三种DNA形式（超螺旋、带切口和线性）随时间的浓度变化进行了同步分析。结果表明，表征酶与超螺旋DNA结合的速率常数（6.0×10⁵ M⁻¹ s⁻¹）和与带切口DNA结合的速率常数（2.8×10⁵ M⁻¹ s⁻¹）在数量级上相似，表征切割第一个（1.2×10⁻² s⁻¹）和第二个磷酸二酯键的速率常数（3.1×10⁻² s⁻¹）也相似。该分析得出带切口中间体从酶上解离的动力学测定平衡常数为12.9 nM。描述带切口中间体从酶上释放的速率常数的值为3.7×10⁻³ s⁻¹。通过将这个释放速率常数的值与描述第二次切割事件的常数的值进行比较，可以确定与酶结合的带切口中间体中只有10%以游离带切口DNA的形式释放，90%的带切口中间体在未释放的情况下被加工成线性形式。因此，大多数DNA是单次酶 - DNA识别事件的结果而被切割。分析过程中未做稳态假设。方法是使用交互式方法直接求解描述动力学过程的微分方程。这项研究证明了这种方法在分析限制性内切核酸酶的蛋白质 - DNA相互作用动力学方面的有用性。