Department of Physics, Nanosystems Initiative Munich, and Center for Nanoscience, LMU Munich, 80799 Munich, Germany.
Department of Biochemistry, Stanford University, Stanford, CA 94305, USA.
Nucleic Acids Res. 2020 Aug 20;48(14):8090-8098. doi: 10.1093/nar/gkaa509.
The DNA four-way (Holliday) junction is the central intermediate of genetic recombination, yet key aspects of its conformational and thermodynamic properties remain unclear. While multiple experimental approaches have been used to characterize the canonical X-shape conformers under specific ionic conditions, the complete conformational ensemble of this motif, especially at low ionic conditions, remains largely undetermined. In line with previous studies, our single-molecule Förster resonance energy transfer (smFRET) measurements of junction dynamics revealed transitions between two states under high salt conditions, but smFRET could not determine whether there are fast and unresolvable transitions between distinct conformations or a broad ensemble of related states under low and intermediate salt conditions. We therefore used an emerging technique, X-ray scattering interferometry (XSI), to directly probe the conformational ensemble of the Holliday junction across a wide range of ionic conditions. Our results demonstrated that the four-way junction adopts an out-of-plane geometry under low ionic conditions and revealed a conformational state at intermediate ionic conditions previously undetected by other methods. Our results provide critical information to build toward a full description of the conformational landscape of the Holliday junction and underscore the utility of XSI for probing conformational ensembles under a wide range of solution conditions.
DNA 四链（Holliday）连接点是遗传重组的核心中间产物，但它的构象和热力学性质的关键方面仍不清楚。虽然已经有多种实验方法被用于在特定离子条件下表征典型的 X 形构象，但该结构的完整构象集合，特别是在低盐条件下，仍在很大程度上未被确定。与先前的研究一致，我们对连接点动力学的单分子Förster 共振能量转移（smFRET）测量揭示了在高盐条件下两个状态之间的转变，但 smFRET 无法确定在低盐和中等盐条件下是否存在快速且无法分辨的不同构象之间的转变，或者是相关状态的广泛集合。因此，我们使用一种新兴技术，X 射线散射干涉测量（XSI），直接探测 Holliday 连接点在广泛的离子条件下的构象集合。我们的结果表明，在低盐条件下，四链连接点采用平面外几何形状，并揭示了一种在中等离子条件下的构象状态，这是以前其他方法未检测到的。我们的结果提供了关键信息，有助于构建 Holliday 连接点构象景观的完整描述，并强调了 XSI 在广泛的溶液条件下探测构象集合的实用性。
