White Rose Research Studentships |2021-2022 Scholarships Grants

Session 2013-2014 â Closing Date Extended to 30 August 2013

Protein-DNA interactions are central to many major cellular processes, including transcription, replication, and packaging of DNA into chromatin. Basic physical parameters of protein-DNA interactions include binding location, association and dissociation rate constants, and in some cases catalytic rate constants. New methods are required to provide a robust and quantitative measure of these important parameters. The project will exploit novel single molecule approaches to examine protein-DNA complexes with unprecedented molecular resolution. The ability to control, manipulate and interrogate single molecules at molecular resolution is a crucial enabling technology that will provide powerful insight into the function of these systems and will aid in the design of new tools to engineer functional devices. Access to a novel enabling technology to characterise the stability, flexibility and function of protein-DNA complexes (force clamp AFM) together with the unique collection of skills and experience of the research team gives this project unrivalled ability.

Objectives

This project will develop quantitative physical approaches to directly measure the stability and binding affinity of protein-DNA complexes that avoid the limitations of ensemble approaches to the study of stochastic processes. We aim to develop the enabling technology and theoretical basis to demonstrate the site-specific single-molecule characterization of the activation energy barrier for the disruption of a protein-DNA binding complex. We will achieve this new capability by combining force-clamp spectroscopy using a custom built instrument and designed force protocols using in-house software. We will characterise the interaction of RecA with single-stranded DNA, the paradigmatic strand exchange complex which is central to recombination and the maintenance of genome stability. Importantly, this enzyme will serve as a model system with which to develop the single molecule experimental tools and analysis, providing a platform for future studies. We will analyse both monomer RecA and also an artificial RecA multimer characterised previously in structural studies. The mechanical properties of the RecA protein both with and without complexed DNA will be analysed, thus investigating the relative importance of protein-DNA interactions in stabilisation of protein structure.