Maurer Jack, Albrecht Claire S, von Hippel Peter H, Marcus Andrew H
Center for Optical, Molecular and Quantum Science, University of Oregon, Eugene, OR 97403, USA.
Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA.
Nucleic Acids Res. 2025 Jan 24;53(3). doi: 10.1093/nar/gkaf047.
Single-stranded-double-stranded DNA (ss-dsDNA) replication forks and primer-template junctions are important recognition sites for the assembly and function of proteins involved in DNA replication, recombination, and repair. DNA 'breathing' - i.e. thermally induced local fluctuations of the sugar-phosphate backbones and bases - can populate metastable conformational macrostates at positions near such junctions and likely play key roles in the functional interactions of the regulatory proteins that bind at these sites. Recently, Maurer et al. [1] performed polarization-sweep single-molecule fluorescence (PS-SMF) studies on exciton-coupled (iCy3)2 dimer-labeled ss-dsDNA fork constructs, which revealed that the nucleobases and backbones immediately adjacent to the dimer probes undergo conformational fluctuations on time scales ranging from hundreds of microseconds to hundreds of milliseconds. The local conformations sensed by the dimer probes consist of four quasi-stable macrostates whose populations and dynamics depend on dimer probe position relative to these junctions. Here we present theoretical analyses of these PS-SMF data that quantify the relative stabilities and activation barriers of the free energy surfaces of site-specific DNA 'breathing' events at key positions within these junctions. Our results suggest a detailed molecular picture for DNA 'breathing' at these positions, thus providing insights into understanding the molecular mechanisms of the proteins that operate at these sites.
单链-双链DNA（ss-dsDNA）复制叉和引物-模板连接点是参与DNA复制、重组和修复的蛋白质组装与功能的重要识别位点。DNA“呼吸”——即糖-磷酸骨架和碱基的热诱导局部波动——可在这些连接点附近的位置形成亚稳态构象宏观状态，并可能在结合于这些位点的调节蛋白的功能相互作用中发挥关键作用。最近，莫勒等人[1]对激子耦合（iCy3）2二聚体标记的ss-dsDNA叉状结构进行了极化扫描单分子荧光（PS-SMF）研究，结果显示，紧邻二聚体探针的核碱基和骨架在数百微秒到数百毫秒的时间尺度上发生构象波动。二聚体探针感知到的局部构象由四种准稳态宏观状态组成，其数量和动力学取决于二聚体探针相对于这些连接点的位置。在此，我们对这些PS-SMF数据进行理论分析，量化这些连接点内关键位置特异性DNA“呼吸”事件自由能表面的相对稳定性和活化能垒。我们的结果揭示了这些位置DNA“呼吸”的详细分子图景，从而为理解在这些位点起作用的蛋白质的分子机制提供了见解。
