Structural classification of NHEJ pathways; unravelling the role of Ku-binding proteins

DNA contains the genetic blueprint for life and allows for cells to be accurately replicated during growth and division. The double-helical string like DNA molecules can however be damaged in multiple ways and this is a fundamental problem for life as alterations to this genetic code can cause cell death and eventually cancer. However, humans have evolved intricate DNA repair systems that can recognise the damage to DNA and act to repair it. DNA damage varies in severity and can be as simple as single base-pair changes or chemical additions, through to breaks across one or both strands of the DNA. A break across both strands of DNA called a DNA-double strand-break (DSBs), is considered to be the most dangerous form of DNA damage as it is the most difficult to repair. Non-homologous end joining (NHEJ), is one of two key mechanisms in humans which repairs DSBs. This mechanism is dependent upon a few core proteins, namely DNA-PKcs, Ku70/80, DNA Ligase IV, XRCC4 and XLF.

Biological molecules such as DNA and the proteins they bind to can be visualised using a few methods, however the proteins involved in NHEJ have typically proven to be difficult to study using these structural biology techniques. Recently however, advances in one such method termed cryo-electron microscopy have allowed us to visualise some of these core proteins and complexes and determine how they interact with each other and DNA. Nevertheless, there are also numerous adaptor proteins involved in NHEJ, that under certain conditions become essential for efficient DNA repair. How these adaptor proteins interact with the core NHEJ machinery is not known. Our recent cryo-EM structures have shown that XLF, a core NHEJ protein is essential for formation of a large multiprotein complex that is required for efficient DNA repair. We believe it is possible that other NHEJ adaptor proteins may also play important roles in the formation of large NHEJ assemblies, and to support this idea we have preliminary data that shows the newly identified adaptor protein, PAXX can stabilise an alternative arrangement of NHEJ proteins.

Therefore, this proposal will examine four adaptor proteins and their role in the formation of large protein complexes during NHEJ. Specifically, we will investigate the proteins PAXX, WRN, CYREN and APLF - and visualise these when they bind to proteins such as DNA-PKcs and Ku70/80 using cryo-electron microscopy. Understanding how these proteins dictate NHEJ complex assembly will allow us to unravel the multiple steps in this intricate mechanism. Targeting NHEJ with drugs is a well-established strategy used in combination with chemo- and radiotherapy for the treatment of many cancers. However, there is long standing concern over the non-specific NHEJ inhibitors that are currently available. We hope that unravelling what may be an intricate multiple step, multiple pathway process, will allow the design of targeted NHEJ inhibitors that could provide more specific and personalised treatments.

DNA repair mechanisms are vital as DNA double-strand breaks (DSBs) can cause genomic instability, cell death and eventually cancer if they are not repaired. Non-homologous end joining (NHEJ) is one of the two key mechanisms required for DSB repair. NHEJ is dependent on a few canonical proteins, namely the large kinase DNA-PKcs, the hetero-dimer Ku70/80, DNA Ligase IV, X-ray repair cross-complementing protein 4 (XRCC4) and XRCC4-like factor (XLF). There are also myriad NHEJ adaptor proteins, in some cases with seemingly overlapping and redundant functions. However, under specific conditions these adaptor proteins can become essential for efficient DNA repair. Many of these adaptor proteins are known to contain Ku-binding motifs (KBM's) which enable them to interact with the NHEJ machinery via the Ku70/80 heterodimer.

Our work has recently revealed two structurally distinct dimeric forms of DNA-PK (the complex of DNA-PKcs, Ku70/80 and DNA), termed long-range synaptic complexes. The KBM containing protein XLF is essential for formation of one of these dimeric structures. Several other NHEJ adaptor proteins also contain KBM's. These include, WRN (Werner syndrome protein), (APLF Aprataxin-and-PNK-Like Factor), CYREN (Cell cycle regulator of NHEJ) and PAXX (PAralog of XRCC4 and XLF). Knowing that the KBM containing protein XLF has a crucial role in the formation of one form of long-range synaptic complex, it is possible that other NHEJ adaptor proteins also have important roles in regulating formation of NHEJ complexes. This proposal will therefore investigate PAXX, WRN, CYREN and APLF in the context of long-range synaptic complexes to determine whether adaptor proteins have a role in regulating the NHEJ mechanism. Specifically, we aim to determine whether these adaptor proteins can direct the formation of different assemblies in response to varying types of DNA damage.