Our team investigates the molecular mechanisms that regulate epigenetic processes involved in human diseases, such as cancer and neurodegenerative disorders. To this aim, we develop and apply single-molecule approaches to image the structural dynamics of protein-nucleic acid complexes across scales (from molecules to cells). We are particularly interested in elucidating various mechanistic aspects of chromatin processing and gene expression, such as chromatin structure and remodelling, DNA repair and RNA expression and localisation. Our single-molecule microscopy technologies reveal the structural dynamics of individual molecules, otherwise hidden in ensemble-averaged experiments, thus enabling us to observe key mechanistic intermediates directly, even when short-lived or at low levels.
We currently focus on four main areas:
- DNA repair and chromatin remodelling dynamics: Each human cell contains the equivalent of two meters of DNA packed in a small, micrometre-sized nucleus in the form of chromatin. The DNA in each of those cells experiences tens of thousands of lesions per day resulting from a variety of chemical, mechanical and radiation sources. Unless this damage is repaired, mutations arise that can lead to numerous diseases such as cancer and neurodegenerative disorders (including Alzheimer’s, Huntington’s and Parkinson’s diseases) that are linked with accumulated DNA damage and defective DNA repair. Elegant systems have evolved to act on chromatin to facilitate the repair process. DNA damage repair requires nucleosome remodelling to allow access to the DNA repair machinery. A group of ATP-dependent enzymes that modify chromatin structure are involved in these processes. These complex multi-subunit machines carry out multiple tasks on nucleosomes including chemical modifications, histone exchange and sliding them on DNA in what appears to be a highly coordinated process. SWR1 complex (ySWR1 in yeast, and hSRCAP in humans) is a 1.1 MDa multi-subunit complex that utilizes ATP to replace canonical H2A histones with the Htz1 variant (H2A.Z in mammalian cells). This chromatin remodelling activity is associated with regulation of gene expression in heterochromatin regions of plant and mammal chromosomes and with the cellular response to DNA damage. Despite a large number of genetic, biochemical and structural studies on ySWR1, its detailed exchange mechanism is still unknown. In collaboration with Prof Dale Wigley (Imperial College), we are investigating the molecular mechanism of ATP-dependent replacement of the canonical two H2A histones with the Htz1 variant by the ySWR1 complex. To this aim, we are developing various single-molecule FRET assays to characterize the multi-step DNA unwrapping process required for remodelling.
- Dynamics of SMC complexes: Throughout the life of a eukaryotic cell, chromosomes undergo drastic conformational rearrangements that play essential roles in almost all nuclear processes, including gene expression, DNA repair and cell division. The super-structure of chromatin is regulated by ring-shaped, ATP-dependent molecular motors belonging to the SMC family of protein complexes. In eukaryotic cells, the three types of SMC complexes are cohesin, condensin and SMC5/6. While some of their biological functions have been well described, the molecular mechanism by which these complexes function remains poorly understood. In collaboration with Prof. Luis Aragon (Cell Cycle Group, MRC LMS), we are developing single-molecule approaches to investigate these mechanisms.
- Single-molecule dynamics in live cells: Imaging individual RNA molecules in live cells is key to understanding fundamental cellular processes such as transcription, translation, splicing, transport and decay. To this aim, we are developing bright and photostable Mango RNA aptamers in collaboration with Prof Peter Unrau (Simon Fraser University). More specifically, we have developed stably folding Mango aptamer arrays and demonstrated their ability to image both coding and non-coding RNAs at single molecule resolution without affecting their known localisation patterns. Thanks to the rapid exchange of photobleached dyes, Mango II arrays enable extended imaging times, which in turn benefits super-resolution techniques such as Structured Illumination Microscopy (SIM). In addition, our Mango II arrays are readily compatible with immunostaining, RNA-FISH, and orthogonal labelling by MS2-arrays. Our Mango arrays enable accurate determination of RNA transcription, nuclear export and subcellular localisation. We are currently exploiting this new technology to investigate several aspects of RNA metabolism, gene expression and chromatin structure in mammalian cells.
- CRISPR/Cas9 off-target mechanism: Over the past decade, the application of CRISPR/Cas9 technology has revolutionised genome editing in biological research. Cas9 is a programmable endonuclease routinely used to generate sequence deletions, insertions, and even to regulate gene expression from bacteria to mammals. However, spurious off-target edits have represented a critical barrier to therapeutic applications. The molecular mechanism by which Cas9 binds and cleaves off-targets remains largely unknown, which is a significant problem that hinders the development of new and improved CRISPR/Cas9 systems with high accuracy and efficiency.
Using a combination of single-molecule approaches, traditional biochemistry, structural biology and cutting-edge genomics, we are elucidating the molecular mechanism by which Cas9 discriminates between on- and off-targets. Our goal is to help develop new and improved CRISPR/Cas9 technology to generate accurate and efficient genome editing tools for therapeutic applications.
Belan O, Barroso C, Kaczmarczyk A, Anand R, Federico S, O’Reilly N, Newton MD, Maeots E, Enchev RI, Martinez-Perez E, Rueda DS, Boulton SJ. (2021). Single-molecule analysis reveals cooperative stimulation of Rad51 filament nucleation and growth by mediator proteins. Molecular Cell 81(5):1058-1073.e7. doi: 10.1016/j.molcel.2020.12.020.
Cawte AD, Unrau PJ and Rueda DS. (2020). Live cell imaging of single RNA molecules with fluorogenic Mango II arrays. Nature Communications 11:1-11
Gutierrez-Escribano P, Newton MD, Llauró A, Huber J, Tanasie L, Davy J, Aly I, Aramayo R, Montoya A, Kramer H, Stigler J, Rueda DS, Aragon L. (2020). A conserved ATP-and Scc2/4-dependent activity for cohesin in tethering DNA molecules. Science Advances 5:eaay6804
Newton MD, Taylor BJ, Driessen RP, Roos L, Cvetesic N, Allyjaun S, Lenhard B, Cuomo ME & Rueda DS. (2019). DNA stretching induces Cas9 off-target activity. Nature Structural & Molecular Biology, 26, 185-192.
Willhoft O, Ghoneim M, Lin C-L, Chua EYD, Wilkinson M, Chaban Y, Ayala R, McCormack EA, Ocloo L, Rueda DS, Wigley DB. (2018). Structure and dynamics of the yeast SWR1-nucleosome complex Science 362 (6411), eaat7716. DOI: 10.1126/science.aat7716.
Autour A, C Y Jeng S, D Cawte A, Abdolahzadeh A, Galli A, Panchapakesan SSS, Rueda DS, Ryckelynck M, Unrau PJ. (2018). Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells. Nature Communications 9(1), 656.