title: DNA synapsis through transient tetramerization triggers cleavage by Ecl18kI restriction enzyme.
abstract: To cut DNA at their target sites, restriction enzymes assemble into different oligomeric structures. The Ecl18kI endonuclease in the crystal is arranged as a tetramer made of two dimers each bound to a DNA copy. However, free in solution Ecl18kI is a dimer. To find out whether the Ecl18kI dimer or tetramer represents the functionally important assembly, we generated mutants aimed at disrupting the putative dimer-dimer interface and analysed the functional properties of Ecl18kI and mutant variants. We show by atomic force microscopy that on two-site DNA, Ecl18kI loops out an intervening DNA fragment and forms a tetramer. Using the tethered particle motion technique, we demonstrate that in solution DNA looping is highly dynamic and involves a transient interaction between the two DNA-bound dimers. Furthermore, we show that Ecl18kI cleaves DNA in the synaptic complex much faster than when acting on a single recognition site. Contrary to Ecl18kI, the tetramerization interface mutant R174A binds DNA as a dimer, shows no DNA looping and is virtually inactive. We conclude that Ecl18kI follows the association model for the synaptic complex assembly in which it binds to the target site as a dimer and then associates into a transient tetrameric form to accomplish the cleavage reaction.
authors.affiliation: Institute of Biotechnology, Graiciuno 8, LT-02241, Vilnius, Lithuania.
authors.forename: Mindaugas
authors.initials: M
authors.lastname: Zaremba
authors.forename: Amelia
authors.initials: A
authors.lastname: Owsicka
authors.forename: Gintautas
authors.initials: G
authors.lastname: Tamulaitis
authors.forename: Giedrius
authors.initials: G
authors.lastname: Sasnauskas
authors.forename: Luda S
authors.initials: LS
authors.lastname: Shlyakhtenko
authors.forename: Alexander Y
authors.initials: AY
authors.lastname: Lushnikov
authors.forename: Yuri L
authors.initials: YL
authors.lastname: Lyubchenko
authors.forename: Niels
authors.initials: N
authors.lastname: Laurens
authors.forename: Bram
authors.initials: B
authors.lastname: van den Broek
authors.forename: Gijs J L
authors.initials: GJ
authors.lastname: Wuite
authors.forename: Virginijus
authors.initials: V
authors.lastname: Siksnys
mesh_terms: D004247:DNA / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D053837:DNA Cleavage; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D055503:Protein Multimerization
chemical_list: D004247:DNA; C114636:endodeoxyribonuclease Ecl18kI; D015252:Deoxyribonucleases, Type II Site-Specific