Orchestration of the ubiquitin-dependent DNA damage response by the p97 system

The DNA damage response (DDR) is a highly organised process, which involves the spatial and temporal orchestrated removal and recruitment of proteins, achieved by the use of dynamic posttranslational modifications (PTMs). Ubiquitination has emerged as one of the major PTMs in the DDR after DNA double strand breaks. Intensive studies have been performed to understand the regulation and impact of ubiquitination in the DDR. Regulation of important DDR players such as 53BP1, BRCA1 or RAD51 strictly depend on the signaling cascade initiated by the two main ubiquitin E3 ligases RNF8 and RNF168 at the site of DNA damage. Recently, it has emerged that the AAA ATPase p97 (VCP/Cdc48) plays a crucial role in the ubiquitin axis of the DDR. p97 interacts with a vast set of cofactors that target p97 to specific substrates and assist it in its function as a molecular segregase. However, the exact role of p97 and its cofactors in the response to ionising radiation induced DSBs and ubiquitination is still poorly understood. In this thesis, three p97 cofactors and their role in the DSB-triggered ubiquitination have been studied. The results show that the so-far poorly characterised UBE4A and UBE4B, two E3/E4 ubiquitin enzymes and p97 associated proteins (cofactors), play critical roles in the regulation of ubiquitination at the sites of DNA damage and absence of either affects both K48- and K63-linked ubiquitin chains. Furthermore, another cofactor of p97, the deubiquitinating enzyme Ataxin-3, affects the regulation of RNF8 and RNF168 at the sites of DNA damage. All three proteins, therefore, help to fine-tune the DDR and the recruitment of critical DDR proteins including BRCA1 or 53BP1. Together, the results of this thesis discover and characterise three new players to the ubiquitination axis at the site of DNA damage. Moreover, Ataxin-3 appears to be an interesting druggable target for two-hit radiotherapy for cancer treatment, namely, the inhibition of Ataxin-3 deubiquitinating activity in combination with ionising radiation.