Physical Sciences of Imaging in the Biomedical Sciences CDT
Thesis project - "Single-molecule methods for visualizing protein-DNA interactions"
Dr Rob Neely, School of Chemistry
Dr Iain Styles, School of Computer Science
Professor Stephen Busby, School of Biosciences
This project will investigate the binding and diffusion of DNA binding proteins on single DNA molecules, by developing novel single molecule methods for visualising the DNA sequence in tandem with the proteins that bind to it.
DNA binding proteins that bind to specific regions of DNA do so with remarkable accuracy and speed which forms the foundation for all genetic processes, including in particular transcriptional regulation.
The Diffusion Problem
In fact DNA binding proteins are able to bind far more rapidly to their target sequences than predicted by random diffusion alone.1 Hence the search for the target sequence is said to proceed by ‘facilitated diffusion’, where the protein binds randomly to a non-specific site on the DNA and then uses the DNA to direct its subsequent search. A number of mechanisms have been elucidated, including: one-dimensional sliding or hopping along the DNA; jumping, along the DNA or across DNA strands; and intersegmental transfer involving ‘stepping’ across loops formed in the DNA.
The facilitated diffusion of DNA binding proteins has been well characterised from a theoretical perspective, but many aspects are yet to be examined experimentally. Single molecule techniques provide an ideal way to examine the diffusion of DNA binding proteins and address these questions. Specifically-labelled DNA which allows for sequence specificity has not been used in previous experiments, whilst the stretched molecules of naked DNA that have been used also do not accurately reflect the complex nature of native DNA. It is these sort of imaging challenges that will be addressed by applying novel labelling methods.
As well as the diffusion of DNA-binding proteins it has been shown that transcription is controlled by the structure and location of the genomic DNA. It particular it has been shown by groups in Birmingham that the location of a gene on the E. coli genome will affect its expression2 and that certain locations in the cell may favour gene expression.3 Single molecule techniques can be used to investigate the relationship between DNA binding proteins and the structure and location of DNA during gene regulation.
Single molecule imaging experiments to date have had a number of issues which this project aims to overcome. DNA methyltransferases can be used to label DNA sequence-specifically with, for instance, fluorescent probes. This is a novel technology which allows DNA to be targeted without damage, with high efficiency, and crucially allows sequence context.4 This will allow direct visualisation of the DNA sequence to investigate DNA binding proteins at the single molecule level.
This project will aim to use DNA methyltransferase-directed in both static experiments, such as mapping DNA and the location of bound proteins, and dynamic experiments, imaging the DNA in vitro, by using microfluidics to examine the diffusion and binding of proteins to tethered DNA, or in vivo, by inserting labelled DNA back into cells and imaging.
- Berg, O. G. & von Hippel, P. H. Diffusion-controlled macromolecular interactions. Ann. Rev. Biophys. Bio, 14, 131-160 (1985).
- Bryant, J. A., Sellars, L. E., Busby, S. J. W. & Lee, D. J. Chromosome position effects on gene expression in Escherichia coli K-12. Nucleic Acids Res., 42, 11383-11392 (2014).
- Sanchez-Romero, M. A., Lee, D. J., Sanchez-Moran E. & Busby, S. J. W. Location and dynamics of an active promoter in Escherichia coli K-12.Biochem. J., 441, 481-485 (2012).
- Vranken, C., Deen, J., Dirix, L., Stakenborg, T., Dehaen, W., Leen, V., Hofkens, J. & Neely, R. K. Super-resolution optical DNA mapping via DNA methyltransferase-directed click chemistry. Nucleic Acids Res., 42, e50 (2014).