Defining dynamic protein complexes in DNA repair by non-homologous end-joining

Environmental exposure threats like UV light, ionizing radiation (IR), chemicals in food, drugs and tobacco smoke cause disease principally through damage to DNA. Consequently, biological systems have evolved to minimise or reverse this damage. The most toxic damage is double-strand breakage of DNA, and the repair of such damage is also prone to error, which can lead to hereditary defects such as microcephaly, primordial dwarfism and immune deficiencies, as well as causing cancers. The likelihood of error is lower if DNA breaks are identified quickly so that the ends can be brought together and rejoined correctly. The cellular machinery that brings the broken ends together include the structurally related XRCC4 family proteins, which have well-characterised conventional folded domains at one end, and intrinsically disordered regions (IDRs) at the other. The role for the disordered regions is poorly understood, as they are not detected by many experimental techniques, but they are likely to dynamically form short-lived ordered modules that mediate interactions between the repair machinery components. Identifying which regions become most ordered, what structures they make, and what they interact with is fundamental to understanding how DNA repair functions, and how it is regulated. This importance is highlighted by the fact that inhibition of DNA repair factors can be used to treat cancers in targeted ways e.g. via synthetic lethality, as illustrated for example by PARP inhibitors. Understanding the dynamics and structures of the XRCC4 family of proteins, will allow this component of the repair pathway to be modelled more accurately in computer simulations, and may allow new inhibitors to be developed that target this part of the repair pathway.
The project has three objectives:
O1: Identification of modules of short-lived structure in the disordered regions of XRCC4 family proteins (XRCC4, XLF and PAXX), using our novel approach and nuclear magnetic resonance (NMR) spectroscopy
O2: Defining the consequence of disrupting the structure in these modules using a unique cell-based reporter of DNA damage repair.
O3: Identify interactors for the modules by applying a state-of-the-art photo-crosslinking approach
The techniques to be utilised and developed in this project will be applicable to a wide range of systems, particularly those which involve proteins with intrinsically disordered regions. It is becoming increasingly clear that many cellular processes are located in membrane-less organelles, and IDRs are frequently involved in forming these. The structural information provided will mark a step change towards the amenability of the XRCC4 family proteins for therapeutic targeting, which could be exploited in the treatment of cancers.

DNA double-strand breaks (DSBs) represent the most cytotoxic DNA lesions, as if un- or misrepaired they can lead to cell death or genome instability, and in turn cancer. NHEJ repairs the vast majority of DSBs in human cells, and defects in NHEJ factors are associated with microcephaly, primordial dwarfism and immune deficiencies. NHEJ is also inhibited by certain viruses to maintain the efficacy of their replication via genomic integration, collectively highlighting the importance of NHEJ to human health. Key NHEJ factors include the structurally related XRCC4 family proteins XRCC4, XLF and PAXX, acting at various stages during NHEJ. Their dimers comprise an N-terminal globular head domain followed by an extended coiled-coil and a C-terminal intrinsically disordered region (IDR) of varying length. While the head and coiled-coil regions are well characterised, little is known about the IDRs. This is mostly due to the dynamic nature of the IDRs precluding their structural investigation by conventional methods requiring static structures. Such structural information, however, is critical for understanding how IDRs regulate cellular processes on a molecular level, and how they might be selectively targeted in the future. As a consequence, deliberate targeting of IDRs remains unexploited to date. Here, we will apply a highly interdisciplinary approach based on advanced NMR methods, bioinformatics, genetic code expansion and cell biology, to structurally and functionally define the IDRs of XRCC4 family proteins, uncover their transient interactors, and integrate this information into a holistic and dynamic NHEJ model. With the development of a widely applicable methodology for the structural study of IDRs at its core, the proposal will not only expand our mechanistic understanding of NHEJ, but has potential to pave the way for the future design of precision medicines targeting XRCC4 family protein IDRs and the IDRs of many other attractive drug targets.