Project No. 2433

PRIORITY PROJECT

Primary Supervisor

Prof Neil Kad- University of Kent

Co-Supervisor(s)

Prof Peter McHugh – University of Oxford

Dr Mina Brett Pitt – Lumicks (CASE Partner)

Summary

DNA repair pathways maintain the genome of all organisms.

When these pathways fail, the consequences are detrimental leading to disease and accelerated aging. DNA repair factors are also potential therapeutic targets across a number of diseases (infectious, malignant, degenerative), but remain largely unexploited.

Here, we will utilise Prof. Neil Kad’s (NK) expertise in single-molecule imaging to characterise DNA repair pathways using a Lumicks optical trapping system. To date studies have used single purified protein molecules to study their dynamics in isolation. This is powerful, but does not inform about the concerted multistep reactions that occur in vivo. We intend to bridge this gap using a method known as SMADNE (Single-molecule analysis of DNA-binding proteins from nuclear extracts). SMADNE relies upon the use of extracts derived from cells expressing fluorescently tagged proteins and was developed to follow recruitment of DNA repair proteins to damage (1). To improve this technique, we aim to study proteins expressed from endogenous promoters in the budding yeast Saccharomyces cerevisiae. This will enable us to probe the dynamics and molecular basis of entire repair pathways, more reflective of in vivo biology. PJM has extensive experience of generating cell extracts closely resemble those observed in cells (2), that will underpin this project. The project will focus on a DNA repair pathway where both groups have a long-standing interest; and nucleotide excision repair.

The project would suit a student with a strong mechanistic biology background, a student with training in molecular genetics or a student with a biochemical/molecular biology background. Ultimately, the student must want to see these molecules functioning individually using the cutting-edge optical trapping instrumentation. This C-trap was developed by the industrial; project partner Lumicks, who will continue to support the project and advise on potential applications and intellectual property opportunities.