Yao Yumi Minyi, O'Hagan Michael P, Onoon Karn, Givon Lihee, Hamer-Rogotner Shelly, Salinas Raul, Kessler Naama, Dym Orly, Pipatpolkai Tanadet, Schumacher Maria A, Afek Ariel
Department of Chemical and Structural Biology, Weizmann Institute of Science, Rehovot 7610001, Israel.
Department of Biology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
bioRxiv. 2025 Jul 4:2025.06.30.662289. doi: 10.1101/2025.06.30.662289.
Transcription factors (TFs) bind to specific genomic sites to regulate gene expression. These interactions almost universally require DNA deformation and the accumulation of local mechanical strain within the double helix. As a result, TF-DNA recognition is determined not only by the linear base sequence but also by the spatial alignment of bases and phosphates, as well as their ability to adopt and retain structural deformations. However, the sequence-centric focus of existing studies makes it challenging to directly probe DNA structural determinants and to decouple their impact from alterations in base sequences, limiting our ability to unravel the key factors influencing binding beyond the sequence identity and leaving significant gaps in our understanding of the principles governing TF-DNA recognition. Here, we introduce a high-throughput strategy to perturb TF binding sites without altering their base sequence, enabling systematic investigation of the structural features of DNA that govern TF binding. Our method, PIC-NIC, introduces single-strand breaks (SSBs) at every position within the binding site, selectively disrupting backbone continuity while preserving nucleotide identity, with the resulting effects on TF binding measured quantitatively. Applied to 15 human TFs spanning eight structural classes, and supported by seven high-resolution TF-DNA crystal structures and molecular dynamics simulations, PIC-NIC uncovers discrete backbone positions serving as structural anchor points where nicks can abolish binding, rewire sequence preferences, or even enhance affinity. By decoupling structural and chemical contributions, we demonstrate that DNA mechanics-encoded in backbone geometry and continuity-can independently shape binding specificity beyond the linear code of base identity. These findings shift the paradigm of TF-DNA recognition, establishing the backbone not as a passive scaffold, but as a functional determinant capable of directing regulatory mechanisms through its physical architecture.
转录因子（TFs）与特定的基因组位点结合以调节基因表达。这些相互作用几乎普遍需要DNA变形以及双螺旋内局部机械应变的积累。因此，TF-DNA识别不仅取决于线性碱基序列，还取决于碱基和磷酸基团的空间排列，以及它们采用和保持结构变形的能力。然而，现有研究以序列为中心的重点使得直接探测DNA结构决定因素并将其影响与碱基序列改变解耦具有挑战性，限制了我们揭示影响结合的关键因素的能力，超出了序列同一性，并且在我们对TF-DNA识别调控原则的理解上留下了重大空白。在这里，我们引入了一种高通量策略，在不改变TF结合位点碱基序列的情况下对其进行扰动，从而能够系统地研究控制TF结合的DNA结构特征。我们的方法PIC-NIC在结合位点内的每个位置引入单链断裂（SSB），选择性地破坏主链连续性，同时保留核苷酸同一性，并对由此产生的对TF结合的影响进行定量测量。应用于跨越八个结构类别的15种人类TF，并得到七个高分辨率TF-DNA晶体结构和分子动力学模拟的支持，PIC-NIC揭示了作为结构锚点的离散主链位置，在这些位置处切口可以消除结合、改变序列偏好，甚至增强亲和力。通过将结构和化学贡献解耦，我们证明了编码在主链几何形状和连续性中的DNA力学可以独立于碱基同一性的线性编码塑造结合特异性。这些发现改变了TF-DNA识别的范式，确立了主链不是一个被动的支架，而是一个能够通过其物理结构指导调控机制的功能决定因素。
