C OMMUNICATION Oligomeric Structure Diversity within the GIY-YIG Nuclease Family Elena M. Ibryashkina 1 †, Giedrius Sasnauskas 2 †, Alexander S. Solonin 1 , Marina V. Zakharova 1 ⁎ and Virginijus Siksnys 2 ⁎ 1 Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia 2 Institute of Biotechnology, Graiciuno 8, LT-02241 Vilnius, Lithuania Received 10 November 2008; received in revised form 30 December 2008; accepted 23 January 2009 Available online 30 January 2009 The GIY-YIG nuclease domain has been identified in homing endonu- cleases, DNA repair and recombination enzymes, and restriction endonu- cleases. The Type II restriction enzyme Eco29kI belongs to the GIY-YIG nuclease superfamily and, like most of other family members, including the homing endonuclease I-TevI, is a monomer. It recognizes the palindromic sequence 5′-CCGC/GG-3′ (“/” marks the cleavage position) and cuts it to generate 3′-staggered ends. The Eco29kI monomer, which contains a single active site, either has to nick sequentially individual DNA strands or has to form dimers or even higher-order oligomers upon DNA binding to make a double-strand break at its target site. Here, we provide experimental evi- dence that Eco29kI monomers dimerize on a single cognate DNA molecule forming the catalytically active complex. The mechanism described here for Eco29kI differs from that of Cfr42I isoschisomer, which also belongs to the GIY-YIG family but is functional as a tetramer. This novel mechanism may have implications for the function of homing endonucleases and other enzymes of the GIY-YIG family. © 2009 Elsevier Ltd. All rights reserved. Edited by M. Belfort Keywords: restriction endonuclease; Eco29kI; DNA–protein interaction; protein dimerization; GIY-YIG family Nucleases of the GIY-YIG family are involved in many cellular processes, including DNA repair and recombination, transfer of mobile genetic elements, and restriction of incoming foreign DNA. The con- served GIY-YIG residues that specify the family are part of the compact structural domain of ∼ 100 aa, which serves as a scaffold for the coordination of a divalent metal ion required for catalysis of the phosphodiester bond cleavage. The crystal structure of the catalytic domain of I-TevI, the first of any GIY-YIG endonuclease, revealed an α/β-sandwich architecture with a central three-stranded antipar- allel β-sheet flanked by three helices. 1 The putative catalytic residues are located on a shallow, concave surface and comprise a metal coordination site. Most of the GIY-YIG family enzymes are complex multidomain proteins that perform different func- tions by combining the GIY-YIG catalytic domain with different specificity, targeting, or other do- mains. 2 For example, in UvrC excinuclease, which is a part of the prokaryotic multiprotein nucleotide excision repair complex UvrABC, the GIY-YIG domain is fused to the Endo V nuclease domain. 3 I-TevI intron endonuclease, which has the GIY-YIG nuclease domain combined with the DNA recogni- tion domain, promotes intron transfer by a double- strand cut in the target allele, catalyzed by a homing endonuclease encoded within the mobile element. 4 In I-TevI, the role of the DNA-binding domain is to recognize and bind intronless DNA substrate, positioning the N-terminal catalytic domain such that it is poised to generate a staggered double- strand break. I-TevI, however, interacts with its sub- strate as a monomer and has a single active site located in the GIY-YIG domain. Therefore, in order to generate a double-strand break, it either has to make a transient dimer in the context of a DNA complex, yielding two active sites, one for cleavage of each strand, or has to exploit the dynamic proper- ties of the DNA, for example, through DNA bending to enable second strand cleavage by the single active site. 5 The complex multidomain architecture of GIY- YIG family nucleases, however, hinders detailed *Corresponding authors. E-mail addresses: zmarina@ibpm.pushchino.ru; siksnys@ibt.lt. † E.M.I. and G.S. contributed equally to this work and should be treated as joint first authors. doi:10.1016/j.jmb.2009.01.048 J. Mol. Biol. (2009) 387, 10–16 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2009 Elsevier Ltd. All rights reserved.