Direct Monitoring of Allosteric Recognition of Type IIE Restriction Endonuclease EcoRII

Shuntaro Takahashi,Hisao Matsuno,Hiroyuki Furusawa,Yoshio Okahata

doi:10.1074/jbc.m800334200

Shuntaro Takahashi, Hisao Matsuno + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m800334200

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Abstract

EcoRII is a homodimer with two domains consisting of a DNA-binding N terminus and a catalytic C terminus and recognizes two specific sequences on DNA. It shows a relatively complicated cleavage reaction in bulk solution. After binding to either recognition site, EcoRII cleaves the other recognition site of the same DNA (cis-binding) strand and/or the recognition site of the other DNA (trans-binding) strand. Although it is difficult to separate these two reactions in bulk solution, we could simply obtain the binding and cleavage kinetics of only the cis-binding by following the frequency (mass) changes of a DNA-immobilized quartz-crystal microbalance (QCM) responding to the addition of EcoRII in aqueous solution. We obtained the maximum binding amounts (Deltam(max)), the dissociation constants (K(d)), the binding and dissociation rate constants (k(on) and k(off)), and the catalytic cleavage reaction rate constants (k(cat)) for wild-type EcoRII, the N-terminal-truncated form (EcoRII N-domain), and the mutant derivatives in its C-terminal domain (K263A and R330A). It was determined from the kinetic analyses that the N-domain, which covers the catalytic C-domain in the absence of DNA, preferentially binds to the one DNA recognition site while transforming EcoRII into an active form allosterically, and then the secondary C-domain binds to and cleaves the other recognition site of the DNA strand.

Highlights

Thousands of restriction endonucleases have been discovered far, and classified into several types because of their cleavage reactions shown in Fig. 1A [1,2,3]
EcoRII, which is classified as a type IIE restriction endonuclease, is a homodimer [5, 6]
We used a DNA-immobilized 27-MHz quartzcrystal microbalance (QCM)2 to monitor the allosteric DNA recognition and digestion catalyzed by a Type IIE restriction endonuclease of EcoRII

Summary

Introduction

Thousands of restriction endonucleases have been discovered far, and classified into several types because of their cleavage reactions shown in Fig. 1A [1,2,3]. Because the 1-site DNA was immobilized at 19 Ϯ 1 ng (0.55 Ϯ 0.02 pmol) cmϪ2 on the QCM, the first saturation of curve a in Fig. 4B shows a 1:1 binding of the N-domain to the one recognition site of the DNA.

Results

Conclusion