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.

DNA looping by FokI: the impact of twisting and bending rigidity on protein-induced looping dynamics

“…The protein association rate that we find, 6 ± 1 × 10 6 M −1  s −1 , is a factor of 10 below the rates previously reported by TPM measurements on other REases (14,16). We find a protein dissociation rate of 1.1 ± 0.5 s −1 , and rates for loop capture and loop release on IF190 of 1.0 ± 0.2 s −1 and 0.19 ± 0.01 s −1 , respectively, all independent of protein concentration (Figure 3, N  > 25 DNA molecules).…”

“…Nevertheless, above a particular protein concentration (on the order of the K d for the binding of the protein to a single individual site on the DNA), one of the two sites are occupied most of the time, which reduces the problem to just a single monomeric protein associating with the remaining free recognition site. This was found to be the case for the FokI concentrations used in this study, by observing the equilibrium distribution as described in (16). [The K d for the initial binding FokI to DNA containing a single FokI site is ∼4 nM (24).…”

MADS Domain Transcription Factors Mediate Short-Range DNA Looping That Is Essential for Target Gene Expression in Arabidopsis

“…To investigate whether SEP3 and STK are indeed able to mediate interactions between elements in the VDD promoter region, tethered particle motion (TPM) analysis (Finzi and Dunlap, 2003;Pouget et al, 2004;Nelson et al, 2006;Dunlap et al, 2011) was performed using a VDD promoter fragment of 697 bp containing all three CArG boxes in the same arrangement found in vivo ( Figure 1A). TPM is a powerful, single-molecule technique, which is particularly appropriate to monitor proteininduced DNA conformational changes such as looping, bending, and large-scale compaction (Finzi and Gelles, 1995;Guerra et al, 2007;Zurla et al, 2009;Zaremba et al, 2010).…”

DNA-Endonuclease Complex Dynamics by Simultaneous FRET and Fluorophore Intensity in Evanescent Field

“…However, for optimal catalytic activity, two DNA-bound Ecl18kI dimers must associate into a transient tetramer. In the case of a two-site DNA substrate, simultaneous binding of two enzyme dimers to different target sites and their association into a tetramer through conformational DNA diffusion results in a DNA loop (27). Since Ecl18kI is a dimer of two identical subunits and its recognition sequence is symmetric, there can be two different conformations of the protein-DNA complex: the so-called ''parallel'' and ''antiparallel'' loop ( Fig.…”