镁离子在限制性内切核酸酶ApaI的DNA剪切活性中的作用：从单个分子到介观范式的随机动力学。,The Journal of Physical Chemistry B

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镁离子在限制性内切核酸酶ApaI的DNA剪切活性中的作用：从单个分子到介观范式的随机动力学。
The Journal of Physical Chemistry B ( IF 2.8 ) Pub Date : 2021-04-16 , DOI: 10.1021/acs.jpcb.0c10643
Biswajit Das ₁ , Kinshuk Banerjee ₂ , Gautam Gangopadhyay ₁

Affiliation

由于反应物种的拷贝数低，发生在细胞内部的生化反应具有明显的随机特征。限制性核酸内切酶切割DNA的动力学也不例外，正如通过单分子实验确定的那样。在这里，我们提出了一个简单的反应方案，以了解辅酶镁离子在核酸内切酶ApaI作用中的作用。该方法基于裂解产物形成的等待时间分布，这使我们能够通过分析和数值确定相应的速率。该理论是在单分子水平上发展起来的，然后推广到细胞内存在的一系列DNA-核酸内切酶复合物的生物学相关案例。理论速率对辅因子浓度曲线与相关的单分子实验数据相匹配，该数据揭示了辅因子结合的正协同作用，并提供了模型参数的可靠估计。此外，确定了一个参数范围，其中使用变化系数测得的等待时间分散度显着低于泊松极限，并且在体内镁离子浓度水平。在体内条件下，如此低的分散度可在ApaI强大的DNA剪切活性中发挥作用。

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On the Role of Magnesium Ions in the DNA-Scissoring Activity of the Restriction Endonuclease ApaI: Stochastic Kinetics from a Single Molecule to Mesoscopic Paradigm

Biochemical reactions occurring inside cells have significant stochastic signatures due to the low copy number of reacting species. Kinetics of DNA cleavage by restriction endonucleases are no exception as established by single-molecule experiments. Here, we propose a simple reaction scheme to understand the role of the cofactor magnesium ion in the action of the endonuclease ApaI. The methodology is based on the waiting time distribution of cleavage product formation that enables us to determine the corresponding rate both analytically and numerically. The theory is developed at the single-molecule level and then generalized to the biologically relevant case of a population of DNA–endonuclease complexes present inside a cell. The theoretical rate versus cofactor concentration curve is matched with relevant single-molecule experimental data that reveals positive cooperativity of cofactor binding and provides a reliable estimate of model parameters. Furthermore, a parameter range is identified where the dispersion of the waiting time, measured using the coefficient of variation, is significantly lower than the Poisson limit and becomes minimum at the in vivo magnesium ion concentration level. Such low dispersion can play a role in the robust DNA-scissoring activity of ApaI under in vivo conditions.

更新日期：2021-04-29

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