| Title |
DNA synapsis through transient tetramerization triggers cleavage by Ecl18kI restriction enzyme |
| Authors |
Zaremba, Mindaugas ; Owsicka, Amelia ; Tamulaitis, Gintautas ; Sasnauskas, Giedrius ; Shlyakhtenko, Luda S ; Lushnikov, Alexander Y ; Lyubchenko, Yuri L ; Laurens, Niels ; Broek, Bram van den ; Wuite, Gijs J.L ; Šikšnys, Virginijus |
| DOI |
10.1093/nar/gkq560 |
| Full Text |
|
| Is Part of |
Nucleic acids research.. Oxford : Oxford University Press. 2010, vol. 38, no. 20, p. 7142-7154.. ISSN 0305-1048. eISSN 1362-4962 |
| Abstract [eng] |
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. |
| Published |
Oxford : Oxford University Press |
| Type |
Journal article |
| Language |
English |
| Publication date |
2010 |
| CC license |
|
