Single-molecule visualisation of PARP trapping by PARP inhibitors

PARP抑制剂是用于治疗癌症的药物。顾名思义，它们以PARP蛋白为靶标，PARP蛋白在修复受损的DNA方面发挥着重要作用。PARP抑制剂的疗效与PARP1在DNA断裂部位的捕获有关。我们最近发现，PARP1可以缩合受损和未受损的DNA；酶的辅助因子NAD+可以逆转受损DNA的这种缩合；PARP抑制剂olaparib即使在NAD+存在的情况下也能保持DNA的缩合构象。总而言之，这些发现表明PARP-DNA结合、DNA延伸和抑制剂捕获PARP之间存在复杂的相互作用，可能还有它们的治疗功能。为了解开这些不同的影响，我们需要在不同的构象中，在PARP激活和抑制的不同阶段，将PARP与更长的DNA片段结合在一起。在这个项目中，我们将开发和应用新的原子力显微镜(AFM)方法来可逆地捕获、可视化和研究不同构象中的DNA，在纳米分辨率和水溶液中，总体目标是在(亚)分子细节上可视化和理解PARP结合所引起的构象变化。具体目标如下：优化表面功能化方法以促进AFM成像，同时仍允许相当大的分子灵活性。PARP与DNA结合和溶液中DNA的形状的纳米分辨率成像。提供对PARP诱导的DNA缩合的机制洞察。提供对PARP抑制剂对PARP-DNA相互作用的影响的机制洞察。AFM是一种被广泛用于纳米分辨率DNA成像的物理技术。然而，由于技术原因，它的使用在很大程度上仅限于牢固地附着在固体支撑表面的DNA。在这个项目中，我们将开发在DNA上实现这种可以原位改变其形状和力学的方法，并将其应用于研究药物对DNA-蛋白质结合的影响。通过开发新的方法学来研究纳米级的DNA-蛋白质相互作用，该项目与BBSRC优先发展的生物科学技术相一致。

PARP inhibitors are pharmaceutical drugs used to treat cancers. As indicated by the name, they target PARP proteins, which play an important role in repairing damaged DNA. The efficacy of PARP inhibitors correlates with trapping of PARP1 at the site of a DNA break. We have recently discovered that PARP1 condenses both damaged and undamaged DNA; that the enzyme's co-factor NAD+ can revert this condensation for damaged DNA; and that the PARP inhibitor olaparib acts to retain the DNA in a condensed conformation even in the presence of NAD+.Overall, these findings indicate a complex interplay between PARP-DNA binding, DNA extension and PARP trapping by inhibitors, and presumably their therapeutic function. To disentangle these various effects, we need to resolve PARP bound to longer DNA segments in different conformations, in different stages of PARP activation and inhibition.In this project, we will develop and apply novel atomic force microscopy (AFM) methods to reversibly trap, visualise and study DNA in different conformations with and without bound PARP inhibitors, at nanometre resolution and in aqueous solution, with the overall aim to visualise and understand conformational changes induced by PARP binding with and without inhibitors at (sub)molecular detail.The specific objectives are as follows:Optimisation of surface functionalisation methods to facilitate AFM imaging while still allowing substantial molecular flexibility.Nanometre-resolution imaging of PARP binding to and shaping of DNA in solution.Providing mechanistic insight into PARP-induced DNA condensation.Providing mechanistic insight into the effect of PARP inhibitors on PARP-DNA interactions.AFM is a physical technique that has been widely used to image DNA at nanometre resolution. However, for technical reasons, its use is largely limited to DNA that is firmly attached to a solid support surface. In this project, we will develop methods to achieve such resolution on DNA that can change its shape and mechanics in situ, and apply it to study the effect of pharmaceutical drugs on DNA-protein binding.By the development of new methodology to study DNA-protein interactions at nanometre resolution, this project is aligned to the BBSRC priority of Technology development for the biosciences.