Single Molecule Enzymology Laboratory, The Francis Crick Institute, London NW1 1AT, United Kingdom.
Department of Physics and Astronomy, University College London, London WC1E 6BT, United Kingdom.
Proc Natl Acad Sci U S A. 2023 May 30;120(22):e2214209120. doi: 10.1073/pnas.2214209120. Epub 2023 May 22.
Poly(ADP-ribose) polymerases (PARPs) play key roles in DNA damage repair pathways in eukaryotic cells. Human PARPs 1 and 2 are catalytically activated by damage in the form of both double-strand and single-strand DNA breaks. Recent structural work indicates that PARP2 can also bridge two DNA double-strand breaks (DSBs), revealing a potential role in stabilizing broken DNA ends. In this paper, we have developed a magnetic tweezers-based assay in order to measure the mechanical stability and interaction kinetics of proteins bridging across the two ends of a DNA DSB. We find that PARP2 forms a remarkably stable mechanical link (rupture force ~85 pN) across blunt-end 5'-phosphorylated DSBs and restores torsional continuity allowing DNA supercoiling. We characterize the rupture force for different overhang types and show that PARP2 switches between bridging and end-binding modes depending on whether the break is blunt-ended or has a short 5' or 3' overhang. In contrast, PARP1 was not observed to form a bridging interaction across blunt or short overhang DSBs and competed away PARP2 bridge formation, indicating that it binds stably but without linking together the two broken DNA ends. Our work gives insights into the fundamental mechanisms of PARP1 and PARP2 interactions at double-strand DNA breaks and presents a unique experimental approach to studying DNA DSB repair pathways.
聚（ADP-核糖）聚合酶（PARPs）在真核细胞的 DNA 损伤修复途径中发挥着关键作用。人类 PARPs1 和 2 可被双链和单链 DNA 断裂等形式的损伤催化激活。最近的结构研究表明，PARP2 还可以桥接两个 DNA 双链断裂（DSBs），揭示了其在稳定断裂 DNA 末端方面的潜在作用。在本文中，我们开发了一种基于磁镊的测定法，以测量在 DNA DSB 的两个末端之间桥接的蛋白质的机械稳定性和相互作用动力学。我们发现 PARP2 在 5'-磷酸化的钝端 DSB 之间形成了非常稳定的机械连接（断裂力~85 pN），并恢复了扭转连续性，允许 DNA 超螺旋化。我们对不同突出端类型的断裂力进行了表征，并表明 PARP2 根据断裂是钝端还是具有短的 5'或 3'突出端，在桥接和末端结合模式之间切换。相比之下，PARP1 没有观察到在钝端或短突出端 DSB 上形成桥接相互作用，并且与 PARP2 桥形成竞争，表明它稳定结合但不将两个断裂的 DNA 末端连接在一起。我们的工作深入了解了 PARP1 和 PARP2 在双链 DNA 断裂处相互作用的基本机制，并提出了一种独特的实验方法来研究 DNA DSB 修复途径。
