Molecular insights into ubiquitin remodelling factor p97(VCP) in DNA damage response and genome stability
Lead Research Organisation:
University of Oxford
Department Name: UNLISTED
Abstract
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.
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.
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.
Organisations
- University of Oxford (Lead Research Organisation)
- UNIVERSITY OF OXFORD (Collaboration)
- Danish Cancer Society (Collaboration)
- University of La Laguna (Collaboration)
- Institute of Cancer Research UK (Collaboration)
- National Institutes of Health (NIH) (Collaboration)
- Russian Academy of Sciences (Collaboration)
- Tel Aviv University (Collaboration)
People |
ORCID iD |
Kristijan Ramadan (Principal Investigator) |
Publications
Lessel D
(2014)
Mutations in SPRTN cause early onset hepatocellular carcinoma, genomic instability and progeroid features.
in Nature genetics
Puumalainen, Marjo-Riitta
(2014)
Chromatin retention of DNA damage sensors DDB2 and XPC through loss of p97 segregase causes genotoxicity
Puumalainen MR
(2014)
Chromatin retention of DNA damage sensors DDB2 and XPC through loss of p97 segregase causes genotoxicity.
in Nature communications
Lopez-Mosqueda J
(2016)
SPRTN is a mammalian DNA-binding metalloprotease that resolves DNA-protein crosslinks
in eLife
Zimmer J
(2016)
Targeting BRCA1 and BRCA2 Deficiencies with G-Quadruplex-Interacting Compounds.
in Molecular cell
Vaz B
(2016)
Metalloprotease SPRTN/DVC1 Orchestrates Replication-Coupled DNA-Protein Crosslink Repair.
in Molecular cell
Gill M
(2016)
A ruthenium polypyridyl intercalator stalls DNA replication forks, radiosensitizes human cancer cells and is enhanced by Chk1 inhibition
in Scientific Reports
Franz A
(2016)
Chromatin-associated degradation is defined by UBXN-3/FAF1 to safeguard DNA replication fork progression.
in Nature communications
Ramadan K
(2017)
Strategic role of the ubiquitin-dependent segregase p97 (VCP or Cdc48) in DNA replication.
in Chromosoma
Torrecilla I
(2017)
The role of ubiquitin-dependent segregase p97 (VCP or Cdc48) in chromatin dynamics after DNA double strand breaks.
in Philosophical transactions of the Royal Society of London. Series B, Biological sciences
Vaz B
(2017)
DNA-Protein Crosslink Proteolysis Repair.
in Trends in biochemical sciences
Ramadan K
(2017)
Editorial: Ubiquitin and Ubiquitin-Relative SUMO in DNA Damage Response.
in Frontiers in genetics
Hanna GG
(2018)
UK Consensus on Normal Tissue Dose Constraints for Stereotactic Radiotherapy.
in Clinical oncology (Royal College of Radiologists (Great Britain))
Baranes-Bachar K
(2018)
The Ubiquitin E3/E4 Ligase UBE4A Adjusts Protein Ubiquitylation and Accumulation at Sites of DNA Damage, Facilitating Double-Strand Break Repair.
in Molecular cell
McDonald F
(2018)
Oligoprogressive Oncogene-addicted Lung Tumours: Does Stereotactic Body Radiotherapy Have a Role? Introducing the HALT Trial.
in Clinical oncology (Royal College of Radiologists (Great Britain))
Ruggiano A
(2021)
The protease SPRTN and SUMOylation coordinate DNA-protein crosslink repair to prevent genome instability.
in Cell reports
Hume S
(2021)
The NUCKS1-SKP2-p21/p27 axis controls S phase entry.
in Nature communications
Herbert KJ
(2021)
Targeting TOPK sensitises tumour cells to radiation-induced damage by enhancing replication stress.
in Cell death and differentiation
Kilgas S
(2021)
p97/VCP inhibition causes excessive MRE11-dependent DNA end resection promoting cell killing after ionizing radiation
in Cell Reports
Bolland H
(2021)
Links between the unfolded protein response and the DNA damage response in hypoxia: a systematic review.
in Biochemical Society transactions
Ruggiano A
(2021)
The Trinity of SPRTN Protease Regulation.
in Trends in biochemical sciences
Na J
(2021)
SPRTN protease-cleaved MRE11 decreases DNA repair and radiosensitises cancer cells.
in Cell death & disease
Kilgas S
(2021)
Immunofluorescence microscopy-based detection of ssDNA foci by BrdU in mammalian cells.
in STAR protocols
Fielden J
(2022)
TEX264 at the intersection of autophagy and DNA repair.
in Autophagy
Krastev DB
(2022)
The ubiquitin-dependent ATPase p97 removes cytotoxic trapped PARP1 from chromatin.
in Nature cell biology
Description | Breast Cancer Now/Targeting SPRTN protease to overcome PARP inhibitor resistance in breast cancers |
Amount | £199,662 (GBP) |
Funding ID | 2019DecPR1406 |
Organisation | Breast Cancer Now |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 04/2020 |
End | 03/2023 |
Description | Characterisation of SPRTN protease in DNA replication and repair of Top1-cleavage complex |
Amount | £36,000 (GBP) |
Organisation | Nuovo Soldati Foundation for Cancer Research |
Sector | Charity/Non Profit |
Country | Switzerland |
Start | 10/2020 |
End | 09/2023 |
Description | DNA-protein crosslink repair: characterization and regulation of a novel repair machinery. |
Amount | £60,000 (GBP) |
Funding ID | ALTF 1109-2017 |
Organisation | European Molecular Biology Organisation |
Sector | Charity/Non Profit |
Country | Germany |
Start | 08/2018 |
End | 07/2020 |
Description | LAB282 Award |
Amount | £500,000 (GBP) |
Organisation | LAB 282 |
Sector | Charity/Non Profit |
Start | 10/2017 |
End | 09/2020 |
Description | The role of autophagy in reparing chemotherapy-induced DNA lesions in cancer |
Amount | £36,000 (GBP) |
Funding ID | 14548187 |
Organisation | Fonds National de la Recherche (FNR) |
Sector | Charity/Non Profit |
Country | Luxembourg |
Start | 10/2020 |
End | 09/2023 |
Title | ATX3 variants inducible cell lines |
Description | We have created doxycycline inducible HEK293 cell lines to express various ATX3 variants. |
Type Of Material | Cell line |
Year Produced | 2019 |
Provided To Others? | Yes |
Impact | -one original scientific publication |
URL | https://pubmed.ncbi.nlm.nih.gov/31613024/ |
Title | Ataxin 3-knock out HeLa, HEK293 and U2OS cell lines |
Description | We have created Ataxin 3 knock out in three human cell lines (HeLa, HEK293 and U2OS). |
Type Of Material | Cell line |
Year Produced | 2019 |
Provided To Others? | Yes |
Impact | We have directly demonstrated that Ataxin 3 is essential for DNA repair in response to IR. One original scientific publication. |
URL | https://pubmed.ncbi.nlm.nih.gov/31613024/ |
Title | BrdU assay for ssDNA |
Description | We have improved a method for better visualisation of single stranded DNA fragments in human cells. |
Type Of Material | Technology assay or reagent |
Year Produced | 2021 |
Provided To Others? | Yes |
Impact | - one original research publication - this protocol has also been published |
URL | https://pubmed.ncbi.nlm.nih.gov/34888531/ |
Title | SPRTN haploinsufficient HeLa Cell Line |
Description | We have created a SPRTN haploinsufficient cell line and published this tool in Vaz et al. Mol Cell 2016 |
Type Of Material | Cell line |
Year Produced | 2016 |
Provided To Others? | Yes |
Impact | We have directly demonstrated that inactivation of SPRTN causes formation of endogenous DNA-protein cross links. Other researchers requested this cell line for their work and we provided it by the MTA. |
URL | https://pubmed.ncbi.nlm.nih.gov/27871366/ |
Title | TEX264 knock out HeLa and HEK293 cell lines |
Description | We have created TEX264 knock out HeLa and HEK293 for our research. |
Type Of Material | Cell line |
Year Produced | 2020 |
Provided To Others? | Yes |
Impact | Using this tool we have directly validated our findings in human cells and demonstrated that TEX264 is essential for Top1-cc repair. One original scientific publication. A new project has been awarded based on this discovery. |
URL | https://pubmed.ncbi.nlm.nih.gov/32152270/ |
Description | Andrew N J Tutt |
Organisation | Institute of Cancer Research UK |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We investigated the mechanistic insight of the PARP1-p97 interaction. |
Collaborator Contribution | Prof Tutt team provided mouse and patient breast cancer organoids essential for our research. |
Impact | -original scientific publication |
Start Year | 2020 |
Description | Dr Raimundo Freire; ATX3 in DNA replication and repair |
Organisation | University of La Laguna |
Country | Spain |
Sector | Academic/University |
PI Contribution | We have discovered the deubiquitinase Ataxin 3 (ATX3) in DNA damage response and DNA replication. We are analysing p97 proteome after ionizing radiation. |
Collaborator Contribution | My partner developed ATX3 specific antibodies and purified ATX3 protein. My partner is performing bioinformatic analysis of p97 proteome after ionizing radiation. |
Impact | -Abhay et al., EMBO J, 2019. |
Start Year | 2017 |
Description | Jiri Bartek, PARP1-p97 study |
Organisation | Danish Cancer Society |
Department | Danish Cancer Society Research Center |
Country | Denmark |
Sector | Academic/University |
PI Contribution | We have found that the p97 ATPase removes cytotoxic trapped PARP1 from chromatin. |
Collaborator Contribution | -provided p97 inhibitor CuET |
Impact | -original scientific publication |
Start Year | 2019 |
Description | Prof Christopher J Lord |
Organisation | Institute of Cancer Research UK |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We performed biochemical analysis of PARP1 interaction with the p97 system. |
Collaborator Contribution | My collaborator identify the interaction between PARP1 and the p97 system by mass-spectrometry analysis. |
Impact | -grant award -original scientific publication -connection with other research groups (networking) Yes, this collaboration is multi-disciplinary, between clinical oncologists, clinical scientists and basic scientists. |
Start Year | 2019 |
Description | Prof Dianov; Interplay between base excision repair protein XRCC1 and ALDH2 predicts overall survival in lung and liver cancer patients. |
Organisation | Russian Academy of Sciences |
Department | Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences |
Country | Russian Federation |
Sector | Public |
PI Contribution | My team and I have analysed DNA-protein crosslinks in cells where XRCC1 and ALDH2 were inactivated. We are currently working on the regulation of G1/S checkpoint by the NUCKS1-SKP2 axis. |
Collaborator Contribution | My partner (Prof Dianov) found that low ALDH2 expression levels associated with high XRCC1 expression levels are indicative for a poor overall survival, particularly in lung and liver cancer patients. In addition, they found that Mithramycin A, a XRCC1 expression inhibitor, efficiently kills cancer cells expressing low levels of ALDH2. My partner (Prof Dianov) has identified the NUCKS1-SKP2 axis. |
Impact | Interplay between base excision repair protein XRCC1 and ALDH2 predicts overall survival in lung and liver cancer patients. Chen X, Legrand AJ, Cunniffe S, Hume S, Poletto M, Vaz B, Ramadan K, Yao D, Dianov GL. Cell Oncol (Dordr). 2018 Oct;41(5):527-539. doi: 10.1007/s13402-018-0390-8. Epub 2018 Aug 7. PMID: 30088263 A unified model for the G1/S cell cycle transition. Hume S, Dianov GL, Ramadan K. Nucleic Acids Res. 2020 Dec 16;48(22):12483-12501. doi: 10.1093/nar/gkaa1002. |
Start Year | 2017 |
Description | Prof Dianov; NUCKS1-SKP2-p21/p27 axis controls S phase |
Organisation | University of Oxford |
Department | Department of Oncology |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We have delineated the molecular mechanism of NUCKS1-SKP2-p21/p27 axis and how it controls S phase entry. |
Collaborator Contribution | Prof Dianov initiated this project and helped us to recruit one fully-funded DPhil student. |
Impact | -original scientific publication -review paper -one DPhil student |
Start Year | 2017 |
Description | Prof Geoff S Higgins, TOPK sensitises tumour cells |
Organisation | University of Oxford |
Department | Department of Oncology |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We analysed how TOPK regulates DNA replication. |
Collaborator Contribution | Prof Higgins groups found that targeting TOPK sensitises tumour cells to radiation-induced damage. |
Impact | -two original scientific publications |
Start Year | 2019 |
Description | Prof Kiltie; MRN disassembly |
Organisation | University of Oxford |
Department | Department of Oncology |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We have discovered how MRN complex, the main DNA double strand break nuclease, is disassembled and regulated at sites of DNA double strand break after ionising radiation. |
Collaborator Contribution | Prof Kiltie validated this model in a mouse system and patient materials. |
Impact | -original scientific publication -review publication -one shared DPhil student This is multidisciplinary collaboration between clinical oncologists and basic scientists. |
Start Year | 2017 |
Description | Prof Yosef Shiloh: Investigation of the novel E3 ubiquitin ligases in DNA damage response , Tel Aviv University, Israel |
Organisation | Tel Aviv University |
Country | Israel |
Sector | Academic/University |
PI Contribution | We have characterised the recruitment of two new E3-ubiquitin ligases to sites of DNA lesions. |
Collaborator Contribution | The group of Prof Shiloh has identified two new ubiquitin ligases by high-throughput screen. |
Impact | We have discovered the importance of fine-tuning of the ubiquitin signal at sites of DNA double strand breaks for cell survival. This discovery resulted in one original scientific publication in a prestigious scientific journal, Molecular Cell. |
Start Year | 2017 |
Description | Prof. Vallis; A three-in-one-bullet for oesophageal cancer: replication fork collapse, spindle attachment failure and enhanced radiosensitivity generated by a ruthenium(ii) metallo-intercalator. |
Organisation | University of Oxford |
Department | Department of Oncology |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | My team and I have analysed DNA replication fork progression and elucidated one of the mechanisms how a ruthenium(ii) metallo-intercalator causes DNA replication stress and cell killing. |
Collaborator Contribution | My partners identified ruthenium(ii) metallo-intercalator as a potential chemical that kills human cells. |
Impact | DOI: 10.1039/C7SC03712K |
Start Year | 2017 |
Description | Yves Pommier_p97-PARP1 study |
Organisation | National Institutes of Health (NIH) |
Department | National Cancer Institute (NCI) |
Country | United States |
Sector | Public |
PI Contribution | Elucidating the physical and functional interactions between PARP1 and p97. |
Collaborator Contribution | Prof Pommier's team reconstituted one part of our findings in vitro. |
Impact | -original scientific publication |
Start Year | 2021 |
Description | AMAC-COVID19 talk |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | This was a public talk on COVID-19 origin, pathology and vaccine for the Association of Alumni and Friends of Croatian Universities in the United Kingdom (AMAC/Almae Matris Alumni Croaticae UK). |
Year(s) Of Engagement Activity | 2021 |
URL | https://amacuk.net/godisnja-predavanja |
Description | Public Engagement at UKRI Coffee Morning Webinar |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | To promote my MRC supported research work on Cancer among general public and Macmillan Cancer Support Charity. |
Year(s) Of Engagement Activity | 2020 |
Description | STEM for Britain 2022 |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Policymakers/politicians |
Results and Impact | "STEM for BRITAIN is a major scientific poster competition and exhibition which has been held in Parliament since 1997, and is organised by the Parliamentary & Scientific Committee. Chaired by Stephen Metcalfe MP, its aim is to give members of both Houses of Parliament an insight into the outstanding research work being undertaken in UK universities by early-career researchers". |
Year(s) Of Engagement Activity | 2022 |
URL | https://stemforbritain.org.uk/ |
Description | TV interviews |
Form Of Engagement Activity | A broadcast e.g. TV/radio/film/podcast (other than news/press) |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Other audiences |
Results and Impact | More than 10 TV interviews April 2020 until now on COVID-19 pandemic and vaccine development for Croatian National TV and regional TV (Al Jazeera Balkan). |
Year(s) Of Engagement Activity | 2020,2021 |
URL | https://www.youtube.com/watch?v=o1nS_JLgJsU |