Research
Our research focuses on four main themes: (i) fundamental mechanisms of gene regulation, (ii) molecular mechanisms of topoisomerase enzymes, (iii) development of novel single-molecule methods to study genomic processes, and (iv) design of complex nucleic acid substrates for single-molecule studies. To facilitate this, we employ a range of single-molecule techniques, including optical tweezers and fluorescence imaging, alongside biochemical strategies. We also develop analytical approaches to help interpret our single-molecule data. See below for more details.
Fundamental mechanisms of gene regulation
Understanding how mechanical stress regulates DNA structure
DNA is frequently subjected to mechanical stress in the cell, and this can often result in local changes to the structure of the DNA. It is hypothesized that these structural transitions may serve as a means of regulating gene expression. In order to understand this, a key goal of our research is to unravel how the structure of DNA changes in response to mechanical stress. Towards this goal, we have previously used optical tweezers and fluorescence imaging to reveal how sequence, ionic strength and local topology alter the structure of DNA under both tension and torsional stress.
Unravelling the interplay between DNA structure and DNA-protein interactions
Tuning DNA-protein interactions through local changes in DNA structure (and vice versa) is thought to play a vital role in gene regulation. We seek to understand the mechanisms that underpin this interplay. For example, using combined optical tweezers and fluorescence imaging, we have revealed that unwinding of DNA (which occurs frequently in vivo) reduces the ability of the mitochondrial transcription factor TFAM to slide on DNA. We have also demonstrated that posttranslational modifications of TFAM can alter its ability to compact DNA. Together, this indicates how tuning TFAM-DNA interactions may contribute to gene regulation.
Application of optical tweezers and fluorescence imaging to determine the influence of tension and torsional stress on DNA structure. See also PNAS (2013), Nat. Commun. (2016), PNAS (2019) and J. Phys. Chem. B (2021).
Fluorescence kymograph showing the sliding of a single TFAM protein on DNA. See also PNAS (2019) and Nucleic Acids Res. (2018).
Catalytic cycle of Type 1A topoisomerases (upper), and our experimental strategy to measure enzyme-mediated gate opening on single-stranded DNA (lower). See also Nat. Commun. (2022) and Nucleic Acids Res. (2021).
The molecular mechanisms of topoisomerase enzymes
Topoisomerases are vital enzymes that regulate the topological state of DNA during many genomic processes, including replication and transcription. They are also an important target for anti-bacterial and anti-cancer drugs. Motivated by this, a key focus of the lab is on understanding the molecular mechanisms that underpin the biological function of these enzymes.
Type 1A topoisomerases
Our recent work has focussed on Type 1A topoisomerases. These enzymes bind to, and cleave, regions of single-stranded (ss)DNA to create an enzyme-bridged ‘gate’ through which another DNA strand can pass. Using optical tweezers to measure the force-extension properties of ssDNA, we have revealed that the gate generated in ssDNA by human TopoIIIα (a Type 1A topoisomerase) can adjust its size depending on its local environment. This provides important mechanistic insight into how these enzymes perform their functional roles.
For an overview of the application of single-molecule methods to study Type 1A topoisomerases, see here for our recent review.
Development of novel single-molecule methods
A core pillar of our research involves the development of novel single-molecule approaches in order to provide new mechanistic insight into essential genomic processes.
Generating supercoiled DNA using optical tweezers
Negatively supercoiled (underwound) DNA plays a vital role in many genomic processes. To study this, we have pioneered a method called Optical DNA Supercoiling (ODS) that provides the ability to generate and manipulate single molecules of negatively supercoiled DNA using dual-trap optical tweezers. This unique approach allows the supercoiled molecule to be freely manipulated in space and probed using both force spectroscopy and fluorescence imaging. These features provide a powerful means to obtain detailed insight into the structure of underwound DNA and how this regulates DNA-protein interactions.
Measuring biological forces using intercalator fluorescence
We have developed a method to detect local changes in DNA tension by measuring the fluorescence intensity associated with cyanine intercalator dyes (such as Sytox Orange). This method relies on two phenomena associated with these dyes: (i) a significant force-dependent DNA binding affinity, and (ii) a substantially enhanced fluorescence signal when intercalated. We have applied this method to reveal that stretched and entwined DNA molecules, mimicking entangled DNA structures created during mitosis, can exhibit DNA-DNA interactions.
Schematic showing the principle of Optical DNA Supercoiling: repeated stretch and retract cycles (steps 1-5) allow controlled changes in the DNA linking number. See also PNAS (2019).
Kymograph showing the change in intercalator fluorescence upon sliding one entwined DNA molecule through another. The change in fluorescence can be used to measure the frictional force. See also Nano Lett. (2018).
Synthesis of defined arrays of nucleosome positioning sequences, which can be used to generate arrays of nucleosomes for single-molecule studies. See also Sci. Rep. (2020).
Nucleic acid engineering
An important element of our research involves the design and synthesis of novel nucleic acid constructs in order to facilitate the study of complex genomic processes at the single-molecule level.
Engineering DNA with defined topologies
Cellular DNA is often rotationally constrained and is frequently underwound. To mimic this at the single-molecule level, we have designed end-closed DNA constructs that contain multiple biotin moieties at defined positions. When tethered between optically-trapped beads, these constructs are rotationally constrained and can also be underwound in a controlled manner using our ODS method (described above).
Synthesis of defined nucleosome arrays
In Eukaryotic cells, DNA is typically compacted and organized into chromatin. The core unit of chromatin is the nucleosome, which consists of ~147 base-pairs of DNA wrapped around an octamer of histone proteins. To facilitate the study of chromatin at the single-molecule level, we have developed a novel strategy, based on Gibson Assembly cloning, to engineer DNA constructs that contain nucleosome positioning sequences at defined positions. This allows the generation of chromatin-like nucleosome arrays.
Collaborators
Our recent and current research has involved collaborations with the following labs: Prof. Gijs Wuite (VU Amsterdam), Prof. Erwin Peterman (VU Amsterdam), Prof. Ian Hickson (Copenhagen), Dr Carolyn Suzuki (New Jersey Medical School), Prof. David Rueda (Imperial College London), Dr Alice Pyne (Sheffield) and Prof. John Hartley (UCL).