Xu Longfu, Jin Shikai, Urem Mia, Lee Seung-Joo, Lamers Meindert H, Chen Xun, Wolynes Peter G, Wuite Gijs J L
Department of Physics and Astronomy, and LaserLaB Amsterdam, Vrije Universiteit Amsterdam, De Boelelaan 1081, 1081 HV, Amsterdam, The Netherlands.
Center for Theoretical Biological Physics, Rice University, 6100 Main St, Houston, TX, USA.
Nat Commun. 2025 Aug 12;16(1):7431. doi: 10.1038/s41467-025-62531-1.
Single-stranded DNA-binding proteins (SSBs) protect transiently exposed ssDNA, yet how DNA polymerase (DNAp) displaces them during replication remains unclear. Using single-molecule force spectroscopy, dual-color imaging, and molecular dynamics simulations on bacteriophage T7 DNAp and SSB, we investigated molecular mechanisms underlying SSB displacement. T7 SSB modulates replication in a force-dependent manner: enhancing it at low tension by preventing secondary structures while impeding it at high tension. Dual-color imaging shows SSBs remain stationary as DNAp advances, supporting a sequential displacement model. Molecular dynamics suggests that DNAp actively lowers the SSB dissociation energy barrier through interactions mediated by the SSB C-terminal tail. FRET confirms close protein proximity during encounters. Optimal replication requires SSB saturation of ssDNA, establishing a delicate balance between protection and efficiency. This spatiotemporal coordination between DNAp and SSB is critical for resolving molecular collisions and may represent a general mechanism for resolving molecular collisions, ensuring both processivity and genomic integrity.
单链DNA结合蛋白（SSB）可保护瞬时暴露的单链DNA，但DNA聚合酶（DNAp）在复制过程中如何取代它们仍不清楚。我们利用单分子力谱、双色成像以及对噬菌体T7 DNAp和SSB进行分子动力学模拟，研究了SSB取代的分子机制。T7 SSB以力依赖的方式调节复制：在低张力下通过防止二级结构形成来增强复制，而在高张力下则阻碍复制。双色成像显示，随着DNAp前进，SSB保持静止，支持顺序取代模型。分子动力学表明，DNAp通过由SSB C末端尾巴介导的相互作用，积极降低SSB的解离能垒。荧光共振能量转移（FRET）证实了相遇期间蛋白质紧密相邻。最佳复制需要单链DNA被SSB饱和，这在保护和效率之间建立了微妙的平衡。DNAp和SSB之间的这种时空协调对于解决分子碰撞至关重要，可能代表了解决分子碰撞的一般机制，确保了持续合成能力和基因组完整性。
