Understanding the process by which DNA double-strand breaks are repaired via non-homologous end joining (NHEJ) is vital for the design and precision of future anti-cancer medicines and the optimisation of CRISPR/Cas9-based gene editing. The X-ray repair cross-complementing protein 4 (XRCC4) is a core DNA repair factor known for its importance in NHEJ. As part of the DNA repair process it interacts with other proteins including the core NHEJ factor XLF, implicated in tethering the DNA ends together to promote DNA-end ligation; the nucleoskeleton protein IFFO1, which stabilises the broken ends of the DNA; and the ligase LIG4, which facilitates the final ligation step in NHEJ. XRCC4 also interacts with chains of small ubiquitin-like modifier (SUMO) units, which experiments suggest block the sites where the other proteins interact with XRCC4. In this way SUMO regulates the actions of XRCC4 in non-homologous end-joining, but how exactly it does so remains unknown.
This project will utilise Electron Paramagnetic Resonance (EPR) and Nuclear Magnetic Resonance (NMR) methods combined with biophysical experiments to characterise the structure of the XRCC4:polySUMO complex. EPR pulsed dipolar spectroscopy (PDS) techniques, such as Double Electron Electron Resonance (DEER), will be used to measure distances between spin labels added to the surfaces of the proteins at known sites. This will enable us to probe suspected conformational changes induced by SUMO binding, investigate the possibility of polySUMO chains crosslinking between different binding sites and test the validity of existing models. Results from these experiments will be combined to produce models of the interacting complexes using molecular dynamics simulations and protein-protein docking including experimentally derived restraints to guide the calculations. The models will be validated using molecular biology, biochemistry, state-of-the-art cell biology and microscopy approaches, as well as DNA repair assays.
The research is highly multidisciplinary and will equip the student with a wide range of experimental and theoretical skills, including cloning, protein expression/purification, EPR and NMR spectroscopy measurements/data analysis, computational tools, cell culture and DNA repair assays to probe protein:protein interactions and assess their functional importance for the DNA damage response. Training will be provided in all areas and thus, the project could suit a candidate from either a physical sciences background who is willing to learn biochemical/biology techniques, or a biological sciences background who is interested in learning spectroscopy and computational methods.
Interested students should contact the supervisory team for informal discussions prior to making a formal application.
https://www.research.manchester.ac.uk/portal/christine.schmidt.html
https://www.research.manchester.ac.uk/portal/alice.bowen.html
https://personalpages.manchester.ac.uk/staff/matthew.cliff/
https://www.research.manchester.ac.uk/portal/sam.hay.html
https://www.chemistry.manchester.ac.uk/epr/about/
https://www.mib.manchester.ac.uk/research/facilities/
http://www.mcrc.manchester.ac.uk/
https://www.mib.manchester.ac.uk/
http://www.cruk.manchester.ac.uk/
http://www.christie.nhs.uk/
Entry Requirements
Applicants must have obtained, or be about to obtain, at least an upper second class honours degree (or equivalent) in a relevant subject.
How to Apply
To be considered for this project you MUST submit a formal online application form. Please select EPSRC PhD Programme on the online application form. For information on how to apply for this project, please visit the Faculty of Biology, Medicine and Health Doctoral Academy website (https://www.bmh.manchester.ac.uk/study/research/apply/).
Applications must be submitted by the deadline, as late applications will not be considered. Incomplete applications will not be considered. Please ensure your application is complete and includes all required documentation before submission.
Applicants interested in this project should make direct contact with the Primary Supervisor to arrange to discuss the project further as soon as possible.
Equality, Diversity and Inclusion
Equality, diversity and inclusion is fundamental to the success of The University of Manchester, and is at the heart of all of our activities. The full Equality, diversity and inclusion statement can be found on the website https://www.bmh.manchester.ac.uk/study/research/apply/equality-diversity-inclusion/
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