Molecular insights into ubiquitin remodelling factor p97(VCP) in DNA damage response and genome stability

The realisation of this programme will not only contribute to gaining knowledge about the mechanistic function of p97, its adapter and substrates, in the DDR, but will also have a direct impact on the development of novel small-molecule molecularly-targeted cancer drugs for use in combination therapy. The impact of this knowledge and any new agents will contribute to the scientific literature and influence on-going research both within the Gray Institute, the research community in the wider University, and national and international research.

The clinical impact of identifying p97 adapters and substrates that are involved in DSB repair after ionizing-radiation and/or DNA replication fork progression and stability will be to provide powerful biomarker tools. First the establishment of cancer or genome instability diagnostic biomarkers; a direct example of this is our discovery of C1orf124/DVC1 mutation in genetically uncharacterized WRN-like syndrome. This work also has ramifications of importance for biomarkers for precise cancer prognosis, as well as predictive biomarkers for therapeutic intervention. Thus clinicians will have a range of powerful tools that are able to identify patient groups, understand their cancer progression and predict their treatment outcome.

Technical Summary

The goal of this current proposal is to understand and identify the specific role of p97 and its adapters in DNA replication and repair. In addition, we will elucidate the composition of the p97-complex, namely the p97-adapter interactome and p97-substrate proteome. By identifying this complex, we aim to define new targets using agents directed to the p97 complex, for the future development of combination therapy with either chemotherapy and radiotherapy; improving the efficacy of these modalities in the treatment of cancer.

Our specific hypothesis is that a unique p97-adapter composition regulates the removal of K48-polyubiqutinated substrates from sites of DNA double strand breaks (DSB), and that a similar mechanism regulates progression of DNA replication fork. This hypothesis will be tested through the following objectives:

Objective 1: Identify the p97-adapters composition from the UBX-protein family and determine their function in DSB repair:

i) We will identify the UBX-proteins (all of which are p97 adapters) involved in regulation of K48-ubiquitin signal at sites of DSBs; we will determine whether identified UBX-protein(s) regulate p97 recruitment at sites of DSB; establish whether identified UBX-protein(s) are involved in regulation of p97-K48-polyubiquitinated substrates after genotoxic stress; if p97 and identified UBX-protein(s) form physical complexes on chromatin; and finally, visualize DSB-related UBX-proteins at sites of DSB.

ii) Identified UBX-proteins will then be tested for their specificity in DSB repair.

Objective 2: Determine p97 substrates, on damaged and undamaged chromatin:

We will identify p97 adapters which govern p97 chromatin function, under physiological and genotoxic conditions; categorise the p97 substrates on the chromatin under these conditions; and ascertain which p97 processing factors are responsible for ubiquitination and deubiquitination of different substrates during DNA replication and repair.

Objective 3: Determine the mechanism of p97 function in DNA replication, in particular after DNA replication fork collapse:

We will assess the role of p97 in the regulation of RNF8 in DNA replication and genome stability, with special focus on UBX-family and DVC1 adapters. We will establish the role of RNF8 activity during DNA replication, and how p97 controls this process; the role of 53BP1 in DNA replication stress and how p97-DVC1 complex controls this; determine the p97 adapters involved in these processes and thus the relevance for the maintenance of DNA replication forks and; finally to determine if there are direct physical and functional interactions between RNF8-53BP1-DVC1-p97 in DNA replication and replication-related repair.