Non-homologous end joining (NHEJ) is the preferred pathway for the repair of DNA double-strand breaks in humans. Here we describe three structural aspects of the repair pathway: stages, scaffolds and strings. We discuss the orchestration of DNA repair to guarantee robust and efficient NHEJ. We focus on structural studies over the past two decades, not only using X-ray diffraction, but also increasingly exploiting cryo-EM to investigate the macromolecular assemblies.

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Non-homologous end joining (NHEJ) is one of the two principal damage repair pathways for DNA double-strand breaks in cells. In this review, we give a brief overview of the system including a discussion of the effects of deregulation of NHEJ components in carcinogenesis and resistance to cancer therapy. We then discuss the relevance of targeting NHEJ components pharmacologically as a potential cancer therapy and review previous approaches to orthosteric regulation of NHEJ factors. Given the limited success of previous investigations to develop inhibitors against individual components, we give a brief discussion of the recent advances in computational and structural biology that allow us to explore different targets, with a particular focus on modulating protein–protein interaction interfaces. We illustrate this discussion with three examples showcasing some current approaches to developing protein–protein interaction inhibitors to modulate the assembly of NHEJ multiprotein complexes in space and time.

The RAD51 ATPase polymerises on single-stranded DNA to form nucleoprotein filaments (NPFs) that are critical intermediates in the DNA strand-exchange reactions of Homologous Recombination (HR). ATP binding is important to maintain the NPF in a competent conformation for strand pairing and exchange. Once strand exchange is completed, ATP hydrolysis licenses the filament for disassembly. Here we show using high-resolution cryoEM that the ATP-binding site of the RAD51 NPF contains a second metal ion. In the presence of ATP, the metal ion promotes the local folding of RAD51 into the conformation required for DNA binding. The metal ion is absent in the structure of an ADP-bound RAD51 filament, that rearranges in a conformation incompatible with DNA binding. The presence of the second metal ion explains how RAD51 couples the nucleotide state of the filament to DNA binding. We propose that loss of the second metal ion upon ATP hydrolysis drives RAD51 dissociation from the DNA and weakens filament stability, thus contributing to NPF disassembly, a critical step in the completion of HR.