Fernández-Sierra Mónica, Quiñones Edwin
Department of Chemistry, University of Puerto Rico, Río Piedras Campus, Río Piedras, PR 00936, United States.
Department of Chemistry, University of Puerto Rico, Río Piedras Campus, Río Piedras, PR 00936, United States.
Arch Biochem Biophys. 2015 Mar 15;570:40-6. doi: 10.1016/j.abb.2015.02.012. Epub 2015 Feb 21.
Here we characterize the fluorescence of the YOYO dye as a tool for studying DNA-protein interactions in real time and present two continuous YOYO-based assays for sensitively monitoring the kinetics of DNA digestion by λ-exonuclease and the endonuclease EcoRV. The described assays rely on the different fluorescence intensities between single- and double-stranded DNA-YOYO complexes, allowing straightforward determination of nuclease activity and quantitative determination of reaction products. The assays were also employed to assess the effect of single-stranded DNA-binding proteins on the λ-exonuclease reaction kinetics, showing that the extreme thermostable single-stranded DNA-binding protein (ET-SSB) significantly reduced the reaction rate, while the recombination protein A (RecA) displayed no effect.
在这里，我们将YOYO染料的荧光特性作为实时研究DNA-蛋白质相互作用的一种工具进行了表征，并提出了两种基于YOYO的连续检测方法，用于灵敏地监测λ-外切核酸酶和内切核酸酶EcoRV对DNA的消化动力学。所描述的检测方法依赖于单链和双链DNA-YOYO复合物之间不同的荧光强度，从而能够直接测定核酸酶活性并定量测定反应产物。这些检测方法还被用于评估单链DNA结合蛋白对λ-外切核酸酶反应动力学的影响，结果表明，极端耐热的单链DNA结合蛋白（ET-SSB）显著降低了反应速率，而重组蛋白A（RecA）则没有影响。
