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...

Regeneration and wound healing are vital to tissue homeostasis and organism survival. One of the biggest challenges of today’s science and medicine is finding methods and factors to stimulate these processes in the human body. Effective solutions to promote regenerative responses will accelerate advances in tissue engineering, regenerative medicine, transplantology, and a number of other clinical specialties. In this study, we assessed the potential efficacy of a synthetic hexapeptide, RDKVYR, for the stimulation of tissue repair and wound healing. The hexapeptide is marketed under the name “Imunofan” (IM) as an immunostimulant. IM displayed stability in aqueous solutions, while in plasma it was rapidly bound by albumins. Structural analyses demonstrated the conformational flexibility of the peptide. Tests in human fibroblast and keratinocyte cell lines showed that IM exerted a statistically significant (p < 0.05) pro-proliferative activity (30–40% and 20–50% increase in proliferation of fibroblast and keratinocytes, respectively), revealed no cytotoxicity over a vast range of concentrations (p < 0.05), and had no allergic properties. IM was found to induce significant transcriptional responses, such as enhanced activity of genes involved in active DNA demethylation (p < 0.05) in fibroblasts and activation of genes involved in immune responses, migration, and chemotaxis in adipose-derived stem cells derived from surgery donors. Experiments in a model of ear pinna injury in mice indicated that IM moderately promoted tissue repair (8% in BALB/c and 36% in C57BL/6 in comparison to control).

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