Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, MD, United States.
Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, MD, United States.
Methods Enzymol. 2022;672:143-152. doi: 10.1016/bs.mie.2022.03.061. Epub 2022 Apr 22.
Ring-shaped hexameric helicases are an essential class of enzymes that unwind duplex nucleic acids to support a variety of cellular processes. Because of their critical roles in cells, hexameric helicase dysfunction has been linked to DNA damage and genomic instability. Biochemical characterization of hexameric helicase activity and regulation in vitro is necessary for understanding enzyme function and aiding drug discovery efforts. In this chapter, we describe protocols for characterizing mechanisms of helicase loading, activation, and unwinding using the model replicative hexameric DnaB helicase and its cognate DnaC loading factor from E. coli.
环形六聚体解旋酶是一类重要的酶，它们可以解开双链核酸，支持多种细胞过程。由于它们在细胞中的关键作用，六聚体解旋酶功能障碍与 DNA 损伤和基因组不稳定性有关。在体外对六聚体解旋酶活性和调节的生化特性进行分析，对于理解酶功能和辅助药物发现工作是必要的。在本章中，我们描述了使用模型复制性六聚体 DnaB 解旋酶及其来自大肠杆菌的同源 DnaC 加载因子来表征解旋酶加载、激活和展开机制的方案。
