Restriction Enzyme Ecl18kI-Induced DNA Looping Dynamics by Single-Molecule FRET

Many type II restriction endonucleases require binding of two copies of a recognition site for efficient DNA cleavage. Simultaneous interaction of the enzyme with two DNA sites results in DNA loop formation. It was demonstrated with the tethered particle motion technique that such looping is a dynamic process where a DNA loop is repeatedly formed and disrupted. Here we use a better and in the context of protein-induced DNA looping virtually unexploited strategy of single-molecule Förster resonance energy transfer of surface immobilized biomolecules to quantitatively study the dynamics of Ecl18kI endonuclease-induced DNA looping and determine the rate constants of loop formation and disruption. We show that two DNA-bound Ecl18kI dimers efficiently form a bridging tetramer looping out intervening DNA with a rate that is only a few orders of magnitude lower than the diffusion limited rate. On the other hand, the existence of Ecl18kI tetramer is only transient, and the loop is rapidly disrupted within about 1 s

Analytical gel filtration experiments to determine the MvaI oligomeric state and the stoichiometry of DNA binding for the oligoduplex 1 shown in ()

<p><b>Copyright information:</b></p><p>Taken from "Restriction endonuclease MvaI is a monomer that recognizes its target sequence asymmetrically"</p><p></p><p>Nucleic Acids Research 2007;35(6):2035-2046.</p><p>Published online 7 Mar 2007</p><p>PMCID:PMC1874612.</p><p>© 2007 The Author(s)</p> Elution profiles were recorded simultaneously at 260 and 280 nm and deconvoluted to obtain separate curves for the MvaI (blue) and DNA (red) concentration. () MvaI alone, () DNA alone, () mixture with a 2:1 molar excess of MvaI over DNA, () stoichiometric mixture, () mixture with a 2:1 molar excess of DNA over MvaI, () calibration curve for Superose ™ 12 HR 10/30 column (Amersham Biosciences) with standards from Biorad (vitamin B-12, 1.35 kDa; myoglobin, 17 kDa; ovalbumin, 44kDa; IgG, 150 kDa and thyroglobin, 670 kDa)