Nucleic acids research
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.
DNA, DNA Cleavage, Deoxyribonucleases, Type II Site-Specific, Microscopy, Atomic Force, Models, Molecular, Mutation, Protein Binding, Protein Multimerization
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Zaremba, Mindaugas; Owsicka, Amelia; Tamulaitis, Gintautas; Sasnauskas, Giedrius; Shlyakhtenko, Luda S.; Lushnikov, Alexander Y.; Lyubchenko, Yuri L.; Laurens, Niels; van den Broek, Bram; Wuite, Gijs J.L.; and Siksnys, Virginijus, "DNA synapsis through transient tetramerization triggers cleavage by Ecl18kI restriction enzyme." (2010). Journal Articles: Pharmaceutical Sciences. Paper 12.