Site-specific DNA recombination is the protein-mediated breakage, exchange and rejoining of two double stranded DNA molecules at specific sequences. Site-specific recombinases carry out a wide variety of biologically important functions including: integration and excision of viral genomes, resolutions of bacterial chromosome dimers, and the regulation the expression of some genes (1). Two families of site-specific recombinases exist, named after the amino acid residue that cleaves the DNA backbone: tyrosine and serine.
In collaboration with David Sherratt’s group in Oxford biochemistry, we have been using novel single-molecule fluorescence techniques to study two site-specific recombination proteins: Cre and XerCD. These proteins sequentially exchange pairs of DNA strands to form a Holliday junction intermediate followed by resolution of the Holliday junction to form the recombinant product DNA.
To observe large scale conformational changes of DNA, we have also developed a novel method to use single-molecule fluorescence called Tethered Fluorophore Motion (TFM; Refs. 1,2). Combining this technique with single-molecule FRET has allowed us to study tyrosine recombinases in unprecedented detail (1-3), observing both the large and small scale conformational changes as recombination occurs in real time at the slide surface. Further, we were able to use dual-colour TFM measurements to study the assembly and large-scale movements of DNA translocase FtsK, which activates XerCD-based recombination (4).
(A) Schematic of Cre recombination on surface immobilised DNAs. (B) Expected FRET and TFM observables during each stage of the reaction. (C) Representative single-molecule time trace of Cre recombination. (D) Observables of tethered-fluorophore motion for a DNA samples with a short tether (87 bp, left) and a long tether (3000 bp, right); the long tether leads to a single fluorophore image with a larger width.
- Pinkney JNM, Zawadzki P, Mazuryk J, Arciszewska LK, Sherratt DJ, Kapanidis AN, Capturing reaction paths and intermediates in Cre-loxP recombination using single-molecule fluorescence, PNAS, 2012; 109 (51), pp.20871-20876.
- May PFJ, Pinkney JNM, Zawadzki P, Evans GW, Sherratt DJ, and Kapanidis AN. Tethered Fluorophore Motion: Studying Large DNA Conformational Changes by Single-fluorophore Imaging. Biophys J, 2014; 107, 1205-1216.
- Zawadzki P, May PFJ, Baker RA, Pinkney JNM, Kapanidis AN, Sherratt DJ, and Arciszewska LK, Conformational transitions during FtsK translocase activation of individual XerCD-dif recombination complexes, PNAS, 2013 vol. 110 (43) pp. 17302-17307.
- May PF, Zawadzki P, Sherratt DJ, Kapanidis AN, Arciszewska LK. Assembly, translocation, and activation of XerCD-dif recombination by FtsK translocase analyzed in real-time by FRET and two-color tethered fluorophore motion. PNAS, 2015 doi: 10.1073/pnas.1510814112