Direct observation of DNA rotation during branch migration of Holliday junction DNA by Escherichia coli RuvA-RuvB protein complex
Direct observation of DNA rotation during branch migration of Holliday junction DNA by Escherichia coli RuvA-RuvB protein complex
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DOI：
10.1073/pnas.0600753103
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发表时间：
2006-08-01
影响因子：
11.1
通讯作者：
Harada, Yoshie
中科院分区：
文献类型：
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作者：
Han, Yong-Woon;Tani, Tomomi;Harada, Yoshie
大肠杆菌RuvA-RuvB复合物促进霍利迪连接DNA的分支迁移，霍利迪连接DNA是同源重组的中心中间体。与许多DNA马达蛋白一样，RuvA-RuvB通过驱动DNA的螺旋旋转来促进分支迁移。为了更详细地阐明RuvA-RuvB介导的分支迁移机制，我们通过将珠子连接到十字形DNA的一端（该十字形DNA的另一端固定在玻璃表面上），观察了霍利迪连接分支迁移期间的DNA旋转。当将RuvA、RuvB和ATP加入溶液中时，观察到珠旋转。我们测量了在不同ATP浓度下由RuvA-RuvB介导的分支迁移引起的珠的旋转速率。数据提供了65 μ M的K-m值和1.6转每秒的V-max值，其对应于8.3bp每秒。这种DNA旋转的实时观察不仅使我们能够测量RuvA-RuvB介导的分支迁移的动力学，而且还打开了详细阐明分支迁移机制的可能性。
The Escherichia coli RuvA-RuvB complex promotes branch migration of Holliday junction DNA, which is the central intermediate of homologous recombination. Like many DNA motor proteins, it is suggested that RuvA-RuvB promotes branch migration by driving helical rotation of the DNA. To clarify the RuvA-RuvB-mediated branch migration mechanism in more detail, we observed DNA rotation during Holliday junction branch migration by attaching a bead to one end of cruciform DNA that was fixed to a glass surface at the opposite end. Bead rotation was observed when RuvA, RuvB, and ATP were added to the solution. We measured the rotational rates of the beads caused by RuvA-RuvB-mediated branch migration at various ATP concentrations. The data provided a K-m value of 65 mu M and a V-max value of 1.6 revolutions per second, which corresponds to 8.3 bp per second. This real-time observation of the DNA rotation not only allows us to measure the kinetics of the RuvA-RuvB-mediated branch migration, but also opens the possibility of elucidating the branch migration mechanism in detail.