Microbiologie, Adaptation et Pathogénie, Univ Lyon, Université Claude Bernard Lyon 1, INSA-Lyon, CNRS, UMR5240, 11 Avenue Jean Capelle, F-69621 Villeurbanne, France.
Molecular Innovation Unit, Centre Christophe Mérieux, BioMérieux, 5 rue des Berges, 38024 Grenoble Cedex 01, France.
Biochem Soc Trans. 2019 Oct 31;47(5):1511-1531. doi: 10.1042/BST20190558.
Recombinases are responsible for homologous recombination and maintenance of genome integrity. In Escherichia coli, the recombinase RecA forms a nucleoprotein filament with the ssDNA present at a DNA break and searches for a homologous dsDNA to use as a template for break repair. During the first step of this process, the ssDNA is bound to RecA and stretched into a Watson-Crick base-paired triplet conformation. The RecA nucleoprotein filament also contains ATP and Mg2+, two cofactors required for RecA activity. Then, the complex starts a homology search by interacting with and stretching dsDNA. Thanks to supercoiling, intersegment sampling and RecA clustering, a genome-wide homology search takes place at a relevant metabolic timescale. When a region of homology 8-20 base pairs in length is found and stabilized, DNA strand exchange proceeds, forming a heteroduplex complex that is resolved through a combination of DNA synthesis, ligation and resolution. RecA activities can take place without ATP hydrolysis, but this latter activity is necessary to improve and accelerate the process. Protein flexibility and monomer-monomer interactions are fundamental for RecA activity, which functions cooperatively. A structure/function relationship analysis suggests that the recombinogenic activity can be improved and that recombinases have an inherently large recombination potential. Understanding this relationship is essential for designing RecA derivatives with enhanced activity for biotechnology applications. For example, this protein is a major actor in the recombinase polymerase isothermal amplification (RPA) used in point-of-care diagnostics.
重组酶负责同源重组和维持基因组完整性。在大肠杆菌中，重组酶 RecA 与 DNA 断裂处的单链 DNA 形成核蛋白丝，并搜索同源双链 DNA 作为修复断裂的模板。在这个过程的第一步中，单链 DNA 与 RecA 结合并拉伸成 Watson-Crick 碱基配对的三链构象。RecA 核蛋白丝还含有 ATP 和 Mg2+，这两种是 RecA 活性所必需的辅助因子。然后，该复合物通过与双链 DNA 相互作用和拉伸开始同源搜索。由于超螺旋、片段间采样和 RecA 聚集，在相关的代谢时间尺度上进行全基因组同源搜索。当找到并稳定长度为 8-20 个碱基的同源区域时，DNA 链交换开始进行，形成异源双链复合物，通过 DNA 合成、连接和解析的组合来解决。RecA 活性可以在没有 ATP 水解的情况下发生，但后者的活性对于提高和加速该过程是必要的。蛋白质的灵活性和单体-单体相互作用是 RecA 活性的基础，它是协作发挥作用的。结构/功能关系分析表明，可以提高重组酶的重组活性，并且重组酶具有固有的大重组潜力。理解这种关系对于设计具有增强的生物技术应用活性的 RecA 衍生物至关重要。例如，该蛋白是用于即时诊断的重组酶聚合酶等温扩增 (RPA) 中的主要作用因子。
