Dickman Mark J, Ingleston Stuart M, Sedelnikova Svetlana E, Rafferty John B, Lloyd Robert G, Grasby Jane A, Hornby David P
Transgenomic Research Laboratory, Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, UK.
Eur J Biochem. 2002 Nov;269(22):5492-501. doi: 10.1046/j.1432-1033.2002.03250.x.
The RuvABC resolvasome of Escherichia coli catalyses the resolution of Holliday junctions that arise during genetic recombination and DNA repair. This process involves two key steps: branch migration, catalysed by the RuvB protein that is targeted to the Holliday junction by the structure specific RuvA protein, and resolution, which is catalysed by the RuvC endonuclease. We have quantified the interaction of the RuvA protein with synthetic Holliday junctions and have shown that the binding of the protein is highly structure-specific, and leads to the formation of a complex containing two tetramers of RuvA per Holliday junction. Our data are consistent with two tetramers of RuvA binding to the DNA recombination intermediate in a co-operative manner. Once formed this complex prevents the binding of RuvC to the Holliday junction. However, the formation of a RuvAC complex can be observed following sequential addition of the RuvC and RuvA proteins. Moreover, by examining the DNA recognition properties of a mutant RuvA protein (E55R, D56K) we show that the charge on the central pin is critical for directing the structure-specific binding by RuvA.
大肠杆菌的RuvABC解离体催化遗传重组和DNA修复过程中产生的霍利迪连接体的解离。这个过程涉及两个关键步骤：分支迁移，由RuvB蛋白催化，该蛋白由结构特异性的RuvA蛋白靶向到霍利迪连接体；解离，由RuvC核酸内切酶催化。我们已经对RuvA蛋白与合成霍利迪连接体的相互作用进行了定量，并表明该蛋白的结合具有高度的结构特异性，且导致形成一个每个霍利迪连接体包含两个RuvA四聚体的复合物。我们的数据与两个RuvA四聚体以协同方式结合到DNA重组中间体一致。一旦形成，这个复合物会阻止RuvC与霍利迪连接体结合。然而，在依次添加RuvC和RuvA蛋白后，可以观察到RuvAC复合物的形成。此外，通过研究突变型RuvA蛋白（E55R，D56K）的DNA识别特性，我们表明中央针上的电荷对于指导RuvA进行结构特异性结合至关重要。
