DNA Synapsis Through Transient Tetramerization Triggers Cleavage by Ecl18kI Restriction Enzyme

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2010 Jun 24

PMID 20571089

Citations 19

Authors

Mindaugas Zaremba

Amelia Owsicka

Gintautas Tamulaitis

Giedrius Sasnauskas

Luda S Shlyakhtenko

Alexander Y Lushnikov

Yuri L Lyubchenko

Niels Laurens

Bram van den Broek

Gijs J L Wuite

Virginijus Siksnys

Affiliations

Soon will be listed here.

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.

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