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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Holliday junction dynamics and branch migration: Single-molecule analysis
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Holliday junction dynamics and branch migration: Single-molecule analysis

机译：霍利迪连接动力学和分支迁移：单分子分析

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摘要

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 Mg~(2+) 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. Mg~(2+) 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.
机译：霍利迪交界处（HJ）是各种遗传过程的中心中间体，包括同源和位点特异性重组以及DNA复制。分支迁移允许同源DNA区域之间的交换，但DNA重组这一关键步骤的详细机制尚不清楚。在这里，我们报告了单个分子中分支迁移的直接实时检测。使用适当设计的HJ构建体，我们能够跟踪单分子水平的结分支迁移。分支迁移被检测为逐步随机过程，其整体动力学取决于Mg〜（2+）的浓度。我们开发了一种理论方法来分析HJ分支迁移的机制。数据显示了步骤，其中连接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。在有利的构型（扩展的HJ几何形状）中，分支可以迁移到检测到的几个碱基对上，通常作为一个较大的步骤。 Mg〜（2+）阳离子可稳定折叠构象并阻止分支迁移，其周期比跳变步骤更长。构象翻转和可变碱基对跳跃步骤提供了对涉及HJ的遗传过程的调控机制的见解。

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