Bacterial Chromosome Dynamics
Co-workers: Lidia Arciszewska, Rachel Baker, Stephan Uphoff, Katarzyna Ginda, Katarzyna Zawadzka, Florence Wagner, Pawel Zawadzki, Mathew Stracy, Karthik Rajasekar
Our research is aimed at understanding how DNA replication, repair and chromosome unlinking shape bacterial chromosome organization in the context of the living cell. The research observes where genes and molecular machines are positioned as a cell proceeds through its growth and division cycles, and what happens when normal cellular behaviour is perturbed by different methods. Individual components of DNA organizing and processing machines are studied genetically, structurally and biochemically, and information on their molecular action is integrated into the context of their action in cells. Our quantitative imaging, exploits quantitative widefield and super-resolution microscopy, using PALM and 3D-SIM.Up to three separate green fluorescent protein fusions to DNA binding proteins, which mark specific genes, and molecular machine components can be simultaneously tracked in living cells,
thereby relating the assembly of DNA processing machinery in time and space to gene position within chromosomes. A central part of our research focus on the molecular mechanism of action of the SMC complex, MukBEF, in organizing newly replicated DNA and in facilitating chromosome segregation. Finally, the interplay between MukBEF and TopoIV and MatP in the chromosome unlinking and organization is being studied at the biochemical and cellular levels.
Stephan Uphoff is an independent postdoctoral researcher in the Sherratt lab. He is studying DNA repair and mutagenesis using single-molecule and single-cell microscopy. Visit Stephan's website for more details: http://users.ox.ac.uk/~quee2159/.
- Uphoff S, Lord D, Potvin-Trottier L, Okumus B, Sherratt DJ, Paulsson J (2016) Stochastic activation of a DNA damage response causes cell-to-cell mutation rate variation. Science (in press).
- Nolivos S, Upton AL, Badrinarayanan A, Müller J, Zawadzka K, Wiktor J, Gill A, Arciszewska LK, Nicolas E, Sherratt DJ (2016). MatP regulates the coordinated action of topoisomerase IV and MukBEF in chromosome segregation. Nature Commun. 7:10466
- Zawadzki P, Stracy M, Ginda K, Zawadzka K, Lesterlin C, Kapanidis AN, Sherratt DJ (2015).The Localization and Action of Topoisomerase IV in Escherichia coli Chromosome Segregation Is Coordinated by the SMC Complex, MukBEF. Cell Rep;13:2587-96.
- Moolman MC, Krishnan ST, Kerssemakers JW, van den Berg A, Tulinski P, Depken M, Reyes-Lamothe R, Sherratt DJ, Dekker NH. (2014). Slow unloading leads to DNA-bound β2-sliding clamp accumulation in live Escherichia coli cells. Nat Commun. 5:5820
- May PF, Zawadzki P, Sherratt DJ, Kapanidis AN, Arciszewska LK (2015) Assembly, translocation, and activation of XerCD-dif recombination by FtsK translocase analyzed in real-time by FRET and two-color tethered fluorophore motion. PNAS, 112(37):E5133-41.
- Lesterlin C, Ball G, Schermelleh L, Sherratt DJ. (2014). RecA bundles mediate homology pairing between distant sisters during DNA break repair. Nature. 506:249-53.
Figure 1: Studying the dynamics of the sliding clamp in live E. coli cells using microfluidics.
Figure 3: 3D-SIM of a cell with DSB-induced RecA–GFP bundle .
Figure 4: Real time single-molecule imaging was used to study collisions of translocating FtsK with other DNA bound proteins.
Figure 2: 3D-SIM of FtsZ-GFP rings in Streptococcus pneumoniae.
Figure 5: Studying site-specific recombination using Tethered Fluorophore motion-FRET .