Single-molecule visualisation of PARP trapping by PARP inhibitors

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.