Post-Translational Modifications in the Radiation Damage Response

DNA replication and DNA repair are two essential processes for genome amplification and stability. Defects in these two processes lead to genome instability and various human diseases such as premature aging and cancer. We have recently identified that the ubiquitin dependent AAA ATPase p97 plays an essential role in the regulation of DNA replication and DNA repair. The specificity of p97 in the regulation of DNA replication and DNA repair is governed by p97 cofactors. Now, we aim to identify and characterise the p97 system (p97/VCP + cofactors) involved in the regulation of DNA replication and DNA repair. Our current results demonstrate that mutations in p97 cofactors lead to cancer and premature aging in humans. Further identification and characterisation of the p97 system will help us to understand why mutations in p97 cofactors lead to cancer. We will use biochemical and cell biological approaches to delineate molecular mechanisms of the p97 system in DNA replication and repair. Given that cancer cells divide much faster than the majority of human cells and have altered metabolism, which leads to elevated DNA damage, the p97 system has emerged as an attractive drug target for cancer therapy. This award aims to demonstrate a new concept, which is based on the hypothesis that the survival and chemoresistance of many cancers depends on the p97 system, and will pave the way for identification of potential p97 system inhibitors for cancer therapy.

Technical Summary

Posttranslational modifications (PTMs) including ubiquitination and SUMOylation are essential for the DNA damage response (DDR) and DNA replication. The local accumulation of these proteinaceous PTMs at sites of DNA damage and at sites of DNA replication is essential for accumulation and disassembly of DDR and DNA replication proteins and thus paramount for genome stability. Defects in ubiquitin (Ub) and SUMO signals lead to various human diseases including neurodegeneration, premature aging and cancer. The main objective of the group is to decipher the ubiquitin (Ub) and SUMO signals in DDR after ionising radiation (IR) and in DNA replication. To address this objective we study the central component of the ubiquitin-proteasome system (UPS), the AAA+ ATPase p97 (also known as VCP in mammals or Cdc48 in lower eukaryotes). p97 is the Ub-dependent molecular chaperone that, in association with its cofactors (around 30 are known), controls the rate and dynamics of Ub and SUMO dependent protein turnover and degradation. The p97 system (p97+cofactors) is essential for various processes in chromatin biology. My work has identified the role of p97 associated with chromatin for the first time and opened a new arena of research, namely the removal and degradation of chromatin-associated proteins. The Group’s work has further discovered the essential role of p97 in DNA double strand break (DSB) repair after IR, and a direct link between mutations in a component of the p97 system and accelerated aging and cancer in humans. Moreover, we have just identified that p97 cofactor SPARTAN is a DNA-dependent metalloprotease for repair of DNA-protein crosslinks, under-investigated DNA lesions associated with premature ageing and cancer. Based on these findings, we have evidence that a specialised DNA repair pathway exists, which removes DNA-protein crosslinks in vertebrates. By using quantitative proteomic approaches we have identified three p97 sub-complexes, which associate with chromatin after IR. Now, we aim to use biochemical and cell biological approaches in different human normal and cancer cell lines to characterise the p97 system in genome stability. Specifically, we will focus on: (i) characterisation of SPARTAN enzymatic activity and identification of its substrates, and (ii) characterisation of three p97-chromatin associated sub-complexes and how these sub-complexes orchestrate DNA replication and DNA double strand break repair after IR. As components of both the UPS (proteasome inhibitor bortezomib -Velcade¯) and DDR (PARP inhibitor olaparib - Lynparza¯) are druggable targets and approved cancer therapeutics, investigating the p97 system is of importance to extend our understanding of carcinogenesis and developing novel cancer therapies.