Department of Molecular and Cell Biology, University of Texas at Dallas, 800 West Campbell Road, Richardson, TX 75080, USA.
Nucleic Acids Res. 2012 Aug;40(15):7452-64. doi: 10.1093/nar/gks430. Epub 2012 May 15.
The Cre-recombination system has become an important tool for genetic manipulation of higher organisms and a model for site-specific DNA-recombination mechanisms employed by the λ-Int superfamily of recombinases. We report a novel quantitative approach for characterizing the probability of DNA-loop formation in solution using time-dependent ensemble Förster resonance energy transfer measurements of intra- and inter-molecular Cre-recombination kinetics. Our method uses an innovative technique for incorporating multiple covalent modifications at specific sites in covalently closed DNA. Because the mechanism of Cre recombinase does not conform to a simple kinetic scheme, we employ numerical methods to extract rate constants for fundamental steps that pertain to Cre-mediated loop closure. Cre recombination does not require accessory proteins, DNA supercoiling or particular metal-ion cofactors and is thus a highly flexible system for quantitatively analyzing DNA-loop formation in vitro and in vivo.
Cre 重组系统已成为遗传操作高等生物的重要工具，也是 λ-Int 超家族重组酶所采用的位点特异性 DNA 重组机制的模型。我们报告了一种新颖的定量方法，用于使用时间相关的整体Förster 共振能量转移测量来表征溶液中 DNA 环形成的概率，该方法用于测量分子内和分子间 Cre 重组动力学。我们的方法使用了一种创新技术，可在共价闭合 DNA 的特定位点掺入多个共价修饰。由于 Cre 重组酶的机制不符合简单的动力学方案，因此我们采用数值方法提取与 Cre 介导的环闭合有关的基本步骤的速率常数。Cre 重组不需要辅助蛋白、DNA 超螺旋或特定的金属离子辅因子，因此是体外和体内定量分析 DNA 环形成的高度灵活的系统。
