Poor DNA repair and genome instability is now recognised to be involved not only in cancer, but also in several neurodegenerative diseases that link with aging. DNA is more stable than RNA due to its use of deoxyribonucleotides (dNTPs) instead of ribonucleotides (NTPs) to encode information. However, dNTPs are 10-100fold less abundant in cells than NTPs. Although DNA polymerases prefer dNTPs, discrimination is imperfect. It is estimated that 1 NTP is built into DNA per Kb replicated. Ribonucleotides cause DNA breaks, and threaten genome stability. RNaseH2 fulfils an essential role in removing misincorporated ribonucleotides from the genome. Indeed, RNAseH2 insufficiency causes Aicardi-Goutieres syndrome (AGS) a rare congenital disease. Yeast experiments show that loss of RNAseH2 confers a mutator phenotype, and mouse RNaseH2 mutants are embryonic lethal, showing the importance of this enzyme
We are developing the zebrafish as a model for DNA repair, as such repair plays a central role in many diseases of old age. To model the role of RNaseH2 in vivo, we have recently created a 49bp deletion in the conserved central domain of the RNASeH2a gene, which blocks RNAseH2 function as it is a unique and essential catalytic subunit of the complex. However, to our surprise this mutant is homozygous viable and fertile. This could indicate that alternative repair pathways exist in fish which could be clinically highly interesting. A viable RNaseH2 deficient mutant will allow us to study potential effects of this pathway on age-related diseases
*Characterise RNaseH2 mutants: Analysis of DNA damage under various stresses (eg hydroxyurea to deplete dNTPs). Analysis of adult behaviour and aging markers.
*Test interaction with other DNA-repair pathways: The surprising difference between mouse and zebrafish indicates that alternative repair pathways exist.
* Our data with RNAseH2 / ATM double knockout zebrafish suggest elevated incidence of solid tumours. We will assess the nature of the tumours (benign vs malignant). We propose that in the absence of RNaseH2 (& ATM), topoisomerase 1 can process the misincoportated ribonucleotides, causing gene deletions and increased carcinogenesis. The student will employ established DNA repair assays to get further insight into this mechanism.
*Can HIF signalling protect RNaseH2 mutants? We found that HIF can protect embryos defective in, for instance, BRCA2 and ATM function. We will assess whether chemical activation of HIF can protect RNAseH2 mutants, thereby suggesting a clinical strategy for AGS patients.
We will employ a variety of techniques, focused on zebrafish and probably cell culture (qPCR, immunohistochemistry, is situ hybridisation, CRISPR-related techniques, GFP based fluorescence essays, behavioural analysis).
The use of zebrafish as a whole-organism model for DNA repair is novel. Furthermore, the RNAseH2 mutant is a new mutant which questions our current understanding of one form of DNA repair, By applying the new CRISPRi technology to such a mutant, we can quickly screen through candidate pathways that may provide “backup repair”.
Unit of assessment/ Environment
The PhD will be based within in the Faculty of Science/Department of Biomedical Science. Our department was rated 1st in the 2014 REF for medical and top 5 for biological research. The work extends an existing collaboration between Prof El-Khamisy (Dept of molecular biology and biotechnology) and Dr van Eeden; both will be intensively involved in supervision.
Benefits of being in the DiMeN DTP:
This project is part of the Discovery Medicine North Doctoral Training Partnership (DiMeN DTP), a diverse community of PhD students across the North of England researching the major health problems facing the world today. Our partner institutions (Universities of Leeds, Liverpool, Newcastle and Sheffield) are internationally recognised as centres of research excellence and can offer you access to state-of the-art facilities to deliver high impact research.
We are very proud of our student-centred ethos and committed to supporting you throughout your PhD. As part of the DTP, we offer bespoke training in key skills sought after in early career researchers, as well as opportunities to broaden your career horizons in a range of non-academic sectors.
Being funded by the MRC means you can access additional funding for research placements, international training opportunities or internships in science policy, science communication and beyond. See how our current DiMeN students have benefited from this funding here: http://www.dimen.org.uk/overview/student-profiles/flexible-supplement-awards
Further information on the programme can be found on our website: http://www.dimen.org.uk/
1: Cornelio DA, Sedam HN, Ferrarezi JA, Sampaio NM, Argueso JL. Both R-loop
removal and ribonucleotide excision repair activities of RNase H2 contribute substantially to chromosome stability. DNA Repair (Amst). 2017 Apr;52:110-114.
2: Walker C, El-Khamisy SF. Perturbed autophagy and DNA repair converge to
promote neurodegeneration in amyotrophic lateral sclerosis and dementia. Brain.2018 May 1;141(5):1247-1262.
3: Kim HR, Greenald D, Vettori A, Markham E, Santhakumar K, Argenton F, van Eeden F. Zebrafish as a model for von Hippel Lindau and hypoxia-inducible factor signaling. Methods Cell Biol. 2017;138:497-523.