Visual Biochemistry: watching protein machines at work on single DNA molecules

The replication and maintenance of DNA is orchestrated by many complex protein machines, and the work carried out in the DNA:protein interactions unit at the University of Bristol aims to understand how these machines work. Single molecule visualisation experiments, in which we can literally watch the action of protein machines on individual DNA molecules, hold great promise for the analysis of DNA:protein interactions because they allow quantitative mechanistic analysis without complications associated with traditional "bulk" methods. These approaches have, for example, been instrumental in recent studies of DNA recombination (see reference). In this project there will be two general aims. Firstly, we will improve an established method for imaging quantum dot labelled proteins on flow-stretched DNA molecules that is based on total internal reflection fluorescence (TIRF) microscopy. This will include the implementation of robust surface passivation and orthogonal DNA attachment methods and the inclusion of a switchable multichannel microfluidic system for controlled reagent delivery. Subsequently we will exploit this technology to investigate the mechanisms of DNA binding proteins involved in (a) DNA replication initiation and segregation of sister chromatids, (b) DNA break repair, (c) transcription-coupled DNA repair and (d) degradation of invading foreign DNA.

Students will receive interdisciplinary training in biochemistry (protein/DNA purification and characterisation, DNA:protein interaction analysis, reaction kinetics) and physics/engineering (TIRF-based single molecule imaging, image analysis including the use of particle tracking algorithms, microfluidic flow cell design and construction). The project will be co-supervised by Drs Mark Szczelkun and Nigel Savery of the DNA:protein interactions unit, and Dr Annela Seddon from the School of Physics.