Institute of Biophysics, Ulm University, Albert-Einstein-Allee 11, 89081, Ulm, Germany.
Boehringer Ingelheim, CoC CMC Statistics & Data Science, Birkendorfer Str. 65, 88400, Biberach, Germany.
Nat Commun. 2022 Nov 2;13(1):6569. doi: 10.1038/s41467-022-34148-1.
Single-stranded breaks (SSBs) are the most frequent DNA lesions threatening genomic integrity. A highly kinked DNA structure in complex with human PARP-1 domains led to the proposal that SSB sensing in Eukaryotes relies on dynamics of both the broken DNA double helix and PARP-1's multi-domain organization. Here, we directly probe this process at the single-molecule level. Quantitative smFRET and structural ensemble calculations reveal how PARP-1's N-terminal zinc fingers convert DNA SSBs from a largely unperturbed conformation, via an intermediate state into the highly kinked DNA conformation. Our data suggest an induced fit mechanism via a multi-domain assembly cascade that drives SSB sensing and stimulates an interplay with the scaffold protein XRCC1 orchestrating subsequent DNA repair events. Interestingly, a clinically used PARP-1 inhibitor Niraparib shifts the equilibrium towards the unkinked DNA conformation, whereas the inhibitor EB47 stabilizes the kinked state.
单链断裂（SSBs）是威胁基因组完整性的最常见 DNA 损伤。与人 PARP-1 结构域结合的高度扭曲的 DNA 结构使得人们提出，真核生物中 SSB 的检测依赖于断裂的 DNA 双螺旋和 PARP-1 的多结构域组织的动力学。在这里，我们在单分子水平上直接探测这个过程。定量 smFRET 和结构整体计算揭示了 PARP-1 的 N 端锌指如何将 DNA SSB 从基本上未受干扰的构象，通过中间状态转化为高度扭曲的 DNA 构象。我们的数据表明，一种诱导契合机制通过多结构域组装级联驱动 SSB 检测，并刺激与支架蛋白 XRCC1 的相互作用，从而协调随后的 DNA 修复事件。有趣的是，一种临床上使用的 PARP-1 抑制剂尼拉帕尼将平衡向未扭曲的 DNA 构象转移，而抑制剂 EB47 则稳定了扭曲状态。
