Gibbs Dalton R, Mahmoud Roaa, Kaur Anisa, Dhakal Soma
Department of Chemistry, Virginia Commonwealth University, Richmond, Virginia.
Department of Chemistry, Virginia Commonwealth University, Richmond, Virginia.
Biophys J. 2021 May 18;120(10):1894-1902. doi: 10.1016/j.bpj.2021.03.006. Epub 2021 Mar 16.
The repair of double-stranded DNA breaks via homologous recombination involves a four-way cross-strand intermediate known as Holliday junction (HJ), which is recognized, processed, and resolved by a specific set of proteins. RuvA, a prokaryotic HJ-binding protein, is known to stabilize the square-planar conformation of the HJ, which is otherwise a short-lived intermediate. Despite much progress being made regarding the molecular mechanism of RuvA-HJ interactions, the mechanochemical aspect of this protein-HJ complex is yet to be investigated. Here, we employed an optical-tweezers-based, single-molecule manipulation assay to detect the formation of RuvA-HJ complex and determined its mechanical and thermodynamic properties in a manner that would be impossible with traditional ensemble techniques. We found that the binding of RuvA increases the unfolding force (F) of the HJ by ∼2-fold. Compared with the ΔG of the HJ alone (54 ± 13 kcal/mol), the increased free energy of the RuvA-HJ complex (101 ± 20 kcal/mol) demonstrates that the RuvA protein stabilizes HJs. Interestingly, the protein remains bound to the mechanically melted HJ, facilitating its refolding at an unusually high force when the stretched DNA molecule is relaxed. These results suggest that the RuvA protein not only stabilizes the HJs but also induces refolding of the HJs. The single-molecule platform that we employed here for studying the RuvA-HJ interaction is broadly applicable to study other HJ-binding proteins involved in the critical DNA repair process.
通过同源重组修复双链DNA断裂涉及一种被称为霍利迪连接体（HJ）的四链交叉中间体，它由一组特定的蛋白质识别、加工和解析。RuvA是一种原核HJ结合蛋白，已知它能稳定HJ的平面正方形构象，否则该构象是一种寿命短暂的中间体。尽管在RuvA-HJ相互作用的分子机制方面已经取得了很大进展，但这种蛋白质-HJ复合物的机械化学方面仍有待研究。在这里，我们采用了基于光镊的单分子操纵试验来检测RuvA-HJ复合物的形成，并以传统的整体技术无法实现的方式确定了其机械和热力学性质。我们发现，RuvA的结合使HJ的解链力（F）增加了约2倍。与单独的HJ的ΔG（54±13千卡/摩尔）相比，RuvA-HJ复合物增加的自由能（101±20千卡/摩尔）表明RuvA蛋白稳定了HJ。有趣的是，该蛋白仍然与机械解链的HJ结合，当拉伸的DNA分子松弛时，有助于其在异常高的力作用下重新折叠。这些结果表明，RuvA蛋白不仅稳定了HJ，还诱导了HJ的重新折叠。我们在这里用于研究RuvA-HJ相互作用的单分子平台广泛适用于研究参与关键DNA修复过程的其他HJ结合蛋白。
