Adams D E, West S C
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts, U.K.
J Mol Biol. 1995 Mar 31;247(3):404-17. doi: 10.1006/jmbi.1995.0149.
The RuvA and RuvB proteins of Escherichia coli promote the branch migration of Holliday junctions during genetic recombination and the recombinational repair of damaged DNA. Using a topological assay that measures the underwinding of covalently closed duplex DNA, we find that RuvA and RuvB promote the transient unwinding of relaxed or supercoiled DNA. Detection of unwinding by RuvAB requires the presence of ATP and a non-hydrolysable ATP analogue (ATP gamma S), and was not observed in the presence of ATP or ATP gamma S alone. These results indicate that RuvAB catalyse the unwinding and rewinding of duplex DNA via an intermediate that can be stabilised by the presence a non-hydrolysable cofactor. At elevated concentrations of Mg2+ (12 to 30 mM), which are known to favour RuvB binding to DNA without the need for RuvA, RuvB protein alone promotes DNA unwinding. These results show that RuvB protein, an ATPase that forms hexameric ring structures that encircle the DNA, is directly responsible for the DNA unwinding activity exhibited by RuvAB. From these results, we propose that branch migration of Holliday junctions by RuvAB occurs by the passage of double-stranded DNA through the RuvAB complex, in a reaction coupled to transient DNA unwinding.
大肠杆菌的RuvA和RuvB蛋白在基因重组和受损DNA的重组修复过程中促进Holliday连接体的分支迁移。通过一种测量共价闭合双链DNA解旋的拓扑分析方法，我们发现RuvA和RuvB促进松弛或超螺旋DNA的瞬时解旋。检测RuvAB引起的解旋需要ATP和一种不可水解的ATP类似物（ATPγS）的存在，单独存在ATP或ATPγS时未观察到解旋现象。这些结果表明，RuvAB通过一种可被不可水解辅因子稳定的中间体催化双链DNA的解旋和重新缠绕。在已知有利于RuvB在无需RuvA的情况下与DNA结合的较高Mg2+浓度（12至30 mM）下，单独的RuvB蛋白可促进DNA解旋。这些结果表明，RuvB蛋白作为一种形成环绕DNA的六聚体环状结构的ATP酶，直接负责RuvAB所表现出的DNA解旋活性。基于这些结果，我们提出RuvAB介导的Holliday连接体分支迁移是通过双链DNA穿过RuvAB复合物实现的，该反应与瞬时DNA解旋相偶联。
