Representative active sites and generic mechanisms of DNA cleavage by homing endonuclease families. The HNH and His/Cys box endonucleases contain similar nuclease motifs and active sites, and are thought to be related via divergence from a common ancestor. In those enzyme families, an absolutely conserved active site histidine residue directly deprotonates a water molecule; the ability of the histidine side chain to act as a general base is facilitated by a hydrogen bond to a neighboring carbonyl moiety (usually an asparagine side chain). The GIY-YIG endonucleases use a similar mechanism, with the difference that an active site tyrosine appears to serve a similar role as an activated general base, again to deprotonate the incoming nucleophilic water molecule. In contrast, the PD-(D/E)xK and EDxHD endonucleases display similar active site structural motifs and mechanisms that appear to be similar to previously well-characterized type II restriction endonucleases; in those enzymes a metal-bound water molecule acts as the incoming nucleophile. In these enzymes (corresponding to either the restriction or the homing endonuclease catalysts) the precise number of metal ions employed is often not entirely clear (and hence is represented in the figure either as a single-metal or a two-metal-dependent active site). In each panel of the figure, the most conserved catalytic elements (corresponding to those regions that contain the enzymes’ namesake motifs) are shown in red, and the corresponding secondary structural elements of the catalytic cores are labeled. LH1 and LH2 in the middle panel refer to LAGLIDADG helices 1 and 2 in a monomeric LAGLIDADG homing endonuclease.