Capturing reaction paths and intermediates in Cre- loxP recombination using single-molecule fluorescence

Abstract

Site-specific recombination plays key roles in microbe biology and is exploited extensively to manipulate the genomes of higher organisms. Cre is a well studied site-specific recombinase, responsible for establishment and maintenance of the P1 bacteriophage genome in bacteria. During recombination, Cre forms a synaptic complex between two 34-bp DNA sequences called loxP after which a pair of strand exchanges forms a Holliday junction (HJ) intermediate; HJ isomerization then allows a second pair of strand exchanges and thus formation of the final recombinant product. Despite extensive work on the Cre-loxP system, many of its mechanisms have remained unclear, mainly due to the transient nature of complexes formed and the ensemble averaging inherent to most biochemical work. Here, we address these limitations by introducing tethered fluorophore motion (TFM), a method that monitors large-scale DNA motions through reports of the diffusional freedom of a single fluorophore. We combine TFM with Förster resonance energy transfer (FRET) and simultaneously observe both large-and small-scale conformational changes within single DNA molecules. Using TFM-FRET, we observed individual recombination reactions in real time and analyzed their kinetics. Recombination was initiated predominantly by exchange of the "bottom-strands" of the DNA substrate. In productive complexes we used FRET distributions to infer rapid isomerization of the HJ intermediates and that a rate-limiting step occurs after this isomerization. We also observed two nonproductive synaptic complexes, one of which was structurally distinct from conformations in crystals. After recombination, the product synaptic complex was extremely stable and refractory to subsequent rounds of recombination.tethered fluorophore motion | site-specific DNA recombination | Holliday junction dynamics | alternating laser excitation | protein-DNA interactions S ite-specific DNA recombination is the protein-mediated cleavage, exchange, and rejoining of DNA strands between two duplexes containing a specific sequence. Tyrosine recombinases are involved in the integration and excision of viral genomes, resolution of chromosome dimers, and gene expression (1). This large family of proteins shares a general mechanism of recombination whereby a tetramer of recombinases mediates sequential pairs of strand exchange between two DNA duplexes; the first pair forms a Holliday junction (HJ) intermediate, whereas the second leads to resolution of the HJ to recombinant product (Fig. 1A). DNA cleavage requires no external energy factors, as the bond energy is stored during strand exchange as a covalent protein-DNA linkage.One of the best studied tyrosine recombinases is Cre, a 38 kDa protein of the bacteriophage P1 of Escherichia coli. Cre catalyzes cyclization of the viral genome and resolution of plasmid dimers during replication (2). Cre mediates recombination between 34 bp DNA sequences named loxP (Fig. 1B). Unlike other recombinases [e.g., XerCD (3) and λ Int (4)], Cre does not require accessory...

Single-molecule microscopy of Cre recombination

“…By using the tethered fluor method (TFM), synapsis of the two Crebound loxP recombination sites was found to be rapid and most likely diffusion-limited, as it is in the λ-system. Also in accordance with the previous λ-studies, Pinkney et al (1) find that the stability of Cre-mediated site-specific recombination and its analysis by dual-aspect single-molecule microscopy. (A) Schematic of the site-specific recombination reaction carried out by the tyrosine family of recombinases.…”

“…As an added bonus, the fluorophore used for TFM was also the acceptor for smFRET, and both measurements were made by using only a simple total internal reflection microscope. By marrying the two techniques, the authors are able to determine long-range and subtle DNA structure changes in real time (1).…”

“…1A, i) determines the first pair of DNA strands exchanged (6, 9): "bottom" strands in the Cre reaction. Pinkney et al (1) determine that this directionality results in the formation of a bottom-strandbiased synaptic structure eight times more frequently than formation of the topstrand-biased structure. Intriguingly, the…”

Assembly, translocation, and activation of XerCD- dif recombination by FtsK translocase analyzed in real-time by FRET and two-color tethered fluorophore motion

“…We adapted TFM-FRET (24,33,34) to follow the assembly, translocation, and behavior of FtsK C when it interacts with XerCD. We made a DNA substrate carrying two dif sites separated by a 1-kb spacer (Fig.…”

Optimal Background Estimators in Single-Molecule FRET Microscopy

“…1 a and Materials and Methods section). Another advantage of using profile fitting is that the Gaussian model parameters may contain insightful information about the peak of interest, such the number of molecules within the spot (15), the subpixel localization of the molecule (22,23), or the distance from the fluorophore to the surface of the sample (24). The disadvantage of the PSF estimator is that it relies heavily on the goodness of the fit, which in turn relies on start guesses and constraints on model parameters, the signal/noise ratio of the data, and the presence of molecules close to the molecule of interest.…”