Pandey Manjula, Levin Mikhail K, Patel Smita S
Department of Biochemistry, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, NJ, USA.
Methods Mol Biol. 2010;587:57-83. doi: 10.1007/978-1-60327-355-8_5.
DNA unwinding and polymerization are complex processes involving many intermediate species in the reactions. Our understanding of these processes is limited because the rates of the reactions or the existence of intermediate species is not apparent without specially designed experimental techniques and data analysis procedures. In this chapter we describe how pre-steady state and single-turnover measurements analyzed by model-based methods can be used for estimating the elementary rate constants. Using the hexameric helicase and the DNA polymerase from bacteriophage T7 as model systems, we provide stepwise procedures for measuring the kinetics of the reactions they catalyze based on radioactivity and fluorescence. We also describe analysis of the experimental measurements using publicly available models and software gfit ( http://gfit.sf.net ).
DNA解旋和聚合是复杂的过程，反应中涉及许多中间物种。我们对这些过程的理解有限，因为如果没有专门设计的实验技术和数据分析程序，反应速率或中间物种的存在并不明显。在本章中，我们描述了如何通过基于模型的方法分析预稳态和单轮测量来估计基本速率常数。以噬菌体T7的六聚体解旋酶和DNA聚合酶为模型系统，我们提供了基于放射性和荧光测量它们催化反应动力学的逐步程序。我们还描述了使用公开可用的模型和软件gfit（http://gfit.sf.net）对实验测量进行分析的方法。
