Figure 2

Representative structures of homing endonuclease families and subfamilies. Top: three separate types of catalytic nuclease domains (GIY-YIG, HNH and EDxHD) are found in various phage-encoded homing endonucleases (as well as less commonly in organellar genomes). As illustrated by the structure of full-length HNH endonuclease I-HmuI (middle), these nuclease domains are coupled to elongated DNA-binding regions that are involved in contacts to long target sites. Shown in the insets are crystal structures of the catalytic domains of the GIY-YIG endonuclease I-TevI (left) and the EDxHD endonuclease I-Bth0305I (right). Both of those endonucleases display a general domain organization that is similar to that of I-HmuI: a nuclease cleavage domain tethered to an extended DNA binding region that contains multiple structural motifs. Middle: two closely related types of LAGLIDADG homing endonucleases, corresponding to homodimeric and monomeric enzymes, are encoded within organellar and archaeal genomes. Whereas the homodimeric enzymes can be applied to genome engineering after converting their quaternary organization into an artificial monomeric protein (by tethering the two equivalent domains to one another with a peptide linker) the wild-type monomeric enzymes can be used directly for that purpose. In either case, the N- and C-terminal domains of the protein can be individual engineered and then fused to create highly specific gene targeting proteins. Bottom: His-Cys box endonucleases (which harbor a variant of the HNH active site) and PD-(D/E)xK endonucleases are found in protist and cyanobacterial genomes. Both enzymes are multimers (a homodimer and a tetramer, respectively).