Type II restriction endonuclease R.Eco29kI is a member of the GIY-YIG nuclease superfamily

Abstract

Background: The majority of experimentally determined crystal structures of Type II restriction endonucleases (REases) exhibit a common PD-(D/E)XK fold. Crystal structures have been also determined for single representatives of two other folds: PLD (R.BfiI) and half-pipe (R.PabI), and bioinformatics analyses supported by mutagenesis suggested that some REases belong to the HNH fold. Our previous bioinformatic analysis suggested that REase R.Eco29kI shares sequence similarities with one more unrelated nuclease superfamily, GIY-YIG, however so far no experimental data were available to support this prediction. The determination of a crystal structure of the GIY-YIG domain of homing endonuclease I-TevI provided a template for modeling of R.Eco29kI and prompted us to validate the model experimentally.

Oligomeric Structure Diversity within the GIY-YIG Nuclease Family

“…A similar sigmoidal dependency was reported previously for DNA binding by Eco29kI. 7 Titration of fluorescently labeled DNA with Eco29kI resulted in sigmoidal variation of the fluorescence anisotropy signal that obeyed a simple model of cooperative Eco29kI-DNA interaction akin to Eq. (1).…”

“…Both enzymes are relatively small (Mw ∼ 25 kDa), single-domain proteins that harbor a conserved active-site motif characteristic of GIY-YIG family nucleases. 7,8 Cfr42I and Eco29kI share 32% identical and 32% similar amino acid residues, respectively, but display strikingly different oligomeric structures. The Cfr42I restriction enzyme is a homotetrameric protein that has four active sites and cuts four phosphodiester bonds after binding two DNA molecules.…”

Unusual Target Site Disruption by the Rare-Cutting HNH Restriction Endonuclease PacI

“…The most common catalytic motif, that of the 'PD…(D/E)xK' nuclease superfamily, is the the most wide-spread and best understood (Kosinski et al, 2005). Alternative catalytic motifs, associated with quite different core protein folds, have been identified in many additional restriction endonucleases, including the ‘HNH’ (Cymerman et al, 2006; Jakubauskas et al, 2007; Saravanan et al, 2004) and the ‘GIY-YIG’ (Ibryashkina et al, 2007) motifs (both of which are more commonly associated with mobile homing endonucleases from bacteriophage) (Stoddard, 2005). All three of these catalytic lineages are also found in a much wider variety of enzymes involved in DNA metabolism and modification, including those responsible for DNA repair, recombination and fidelity (Cymerman et al, 2006; Dunin-Horkawicz et al, 2006; Kosinski et al, 2005).…”

Diverse Functions of Restriction-Modification Systems in Addition to Cellular Defense

“…Generally, type II REases are homodimeric or homotetrameric and cleave DNA within or near their target site. These enzymes are highly diverse and are known to utilize at least five types of folds: PD-(D/E)XK, PLD, HNH, GIY-YIG, and halfpipe, e.g., R.EcoRI, R.BfiI, R.KpnI, R.Eco29kI, and R.PabI enzymes, respectively (2,(7)(8)(9)(10). Type II enzymes are the most widely studied and are also extensively utilized nucleases in genetic engineering.…”