title: Restriction enzyme Ecl18kI-induced DNA looping dynamics by single-molecule FRET.
abstract: 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.
authors: Institute of Physics, Center for Physical Sciences and Technology , Savanoriu 231, LT-02300, Vilnius, Lithuania.
authors: Danielis
authors: D
authors: Rutkauskas
authors: Milda
authors: M
authors: Petkelyte
authors: Paulius
authors: P
authors: Naujalis
authors: Giedrius
authors: G
authors: Sasnauskas
authors: Gintautas
authors: G
authors: Tamulaitis
authors: Mindaugas
authors: M
authors: Zaremba
authors: Virginijus
authors: V
authors: Siksnys
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation
chemical_list: D004247:DNA; C114636:endodeoxyribonuclease Ecl18kI; D015252:Deoxyribonucleases, Type II Site-Specific