Karymov Mikhail, Daniel Douglas, Sankey Otto F, Lyubchenko Yuri L
School of Life Sciences, and Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287, USA.
Proc Natl Acad Sci U S A. 2005 Jun 7;102(23):8186-91. doi: 10.1073/pnas.0407210102. Epub 2005 May 25.
The Holliday junction (HJ) is a central intermediate in various genetic processes including homologous and site-specific recombination and DNA replication. Branch migration allows the exchange between homologous DNA regions, but the detailed mechanism for this key step of DNA recombination is unidentified. Here, we report direct real-time detection of branch migration in individual molecules. Using appropriately designed HJ constructs we were able to follow junction branch migration at the single-molecule level. Branch migration is detected as a stepwise random process with the overall kinetics dependent on Mg2+ concentration. We developed a theoretical approach to analyze the mechanism of HJ branch migration. The data show steps in which the junction flips between conformations favorable to branch migration and conformations unfavorable to it. In the favorable conformation (the extended HJ geometry), the branch can migrate over several base pairs detected, usually as a single large step. Mg2+ cations stabilize folded conformations and stall branch migration for a period considerably longer than the hopping step. The conformational flip and the variable base pair hopping step provide insights into the regulatory mechanism of genetic processes involving HJs.
霍利迪连接体（HJ）是包括同源重组、位点特异性重组和DNA复制在内的多种遗传过程中的核心中间体。分支迁移允许同源DNA区域之间进行交换，但DNA重组这一关键步骤的详细机制尚不清楚。在此，我们报告了对单个分子中分支迁移的直接实时检测。使用经过适当设计的HJ构建体，我们能够在单分子水平上跟踪连接体分支迁移。分支迁移被检测为一个逐步随机过程，其整体动力学取决于Mg2+浓度。我们开发了一种理论方法来分析HJ分支迁移的机制。数据显示，连接体在有利于分支迁移的构象和不利于分支迁移的构象之间翻转。在有利构象（扩展的HJ几何结构）中，分支可以跨越检测到的几个碱基对迁移，通常是作为一个大的单步迁移。Mg2+阳离子稳定折叠构象，并使分支迁移停滞一段比跳跃步骤长得多的时间。构象翻转和可变碱基对跳跃步骤为涉及HJs的遗传过程的调控机制提供了见解。
