Institut d'Electronique Microelectronique et Nanotechnologie (IEMN CNRS UMR8520) and Département de Physique, Université de Lille, 59652, Villeneuve d'Ascq, France.
Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, UMR9020-U1277 - CANTHER - Cancer Heterogeneity, Plasticity and Resistance to Therapies, 59000, Lille, France.
Sci Rep. 2024 Oct 6;14(1):23257. doi: 10.1038/s41598-024-73707-y.
The DNA single-strand break (SSB) repair pathway is initiated by the multifunctional enzyme PARP-1, which recognizes the broken DNA ends by its two zinc-finger domains, Zn1 and Zn2. Despite a number of experiments performed with different DNA configurations and reduced fragments of PARP-1, many details of this interaction that is crucial to the correct initiation of the repair chain are still unclear. We performed Molecular Dynamics (MD) computer simulations of the interaction between the Zn1/Zn2 domains of PARP-1 and a DNA hairpin including a missing nucleotide to simulate the presence of an SSB, a construct used in recent experiments. The role of Zn1 and Zn2 interacting with the SSB ends is studied in detail, both independently and cooperatively. We also explored, PARP-1 operating as a dimer, with the two Zn-fingers coming from two separate copies of the enzyme. By an extensive set of all-atom molecular simulations employing state-of-the art force fields, assisted by empirical docking and free-energy calculations, we conclude that the particular conformation of the DNA hairpin can indeed spontaneously open up by thermal fluctuations, up to extremely kinked deformations. However, such extreme localized deformations are rarely observed in free, long DNA fragments. Protein side-loops make contact with the DNA hairpin grooves, and help Zn2 to penetrate deep in the SSB gap. In this way, Zn2 can interact with the nucleotides opposite to the missing base. Overall, Zn1 plays a secondary role: the crucial factor for the interaction is rather the relative arrangement of the Zn1/Zn2 couple, and their mutual orientation with respect to the and SSB end terminals. This helps to obtain an early interacting configuration, which ultimately leads to molecular PARP-1-DNA structures similar to those observed experimentally. Such findings represent an important step toward defining the detailed function of PARP-1 in the early stages of SSB recognition.
DNA 单链断裂 (SSB) 修复途径由多功能酶 PARP-1 启动，该酶通过其两个锌指结构域 Zn1 和 Zn2 识别断裂的 DNA 末端。尽管已经进行了许多使用不同 DNA 构型和 PARP-1 片段的实验，但对于这种相互作用的许多细节，这些细节对于正确启动修复链至关重要，仍然不清楚。我们进行了 PARP-1 的 Zn1/Zn2 结构域与包括缺失核苷酸的 DNA 发夹之间相互作用的分子动力学 (MD) 计算机模拟，以模拟 SSB 的存在，这是最近实验中使用的构建体。详细研究了 Zn1 和 Zn2 与 SSB 末端相互作用的作用，包括独立和协作两种方式。我们还探索了 PARP-1 作为二聚体的作用，其中两个锌指来自酶的两个单独拷贝。通过使用最先进的力场进行广泛的全原子分子模拟，并辅以经验对接和自由能计算，我们得出结论，DNA 发夹的特殊构象确实可以通过热波动自发打开，直到极弯曲的变形。然而，在自由的长 DNA 片段中很少观察到这种极端的局部变形。蛋白质侧环与 DNA 发夹沟道接触，并帮助 Zn2 深入 SSB 缺口。通过这种方式，Zn2 可以与缺失碱基相对的核苷酸相互作用。总体而言，Zn1 发挥次要作用：相互作用的关键因素是 Zn1/Zn2 对的相对排列，以及它们相对于 和 SSB 末端的相对取向。这有助于获得早期相互作用的构型，最终导致与实验观察到的结构相似的分子 PARP-1-DNA 结构。这些发现代表了朝着定义 PARP-1 在 SSB 识别早期阶段的详细功能迈出的重要一步。
