HNH and His Cys Box Homing Endonucleases

The HNH family of endonucleases is characterized by a defining motif that spans 25 residues of a well-conserved sequence. The homing endonucleases I-Cmoel, I-TevIII, I-Hmul, and I-HmuII (Eddy and Gold 1991; Goodrich-Blair and Shub 1996) are members of the HNH family, and I-Cmoel has been well characterized biochemically (Drouin et al. 2000; for a full description of these enzymes, see Keeble et al., this Volume). The non-specific DNase colicin E9 was the first HNH enzyme to have its structure solved (Kleanthous et al. 1999) and allowed biochemical and structural comparisons of the active sites of I-Ppol, Serratia nuclease, colicin E9, and HNH homing endonucleases. These studies led to the hypothesis that these enzymes are evolutionarily related and share similar catalytic mechanisms (Friedhoff et al. 1999; Kuhlmann et al. 1999). Recently, the first structure of an HNH homing endonuclease, I-Hmul, has been solved and confirms these relationships (Shen et al. 2004). The structure of I-Hmul is comprised of an N-terminal antiparallel DNA binding P-sheet (similar to those of the His-Cys box family), the ppa-Me catalytic motif (encoded by the HNH motif), two DNA-binding a-helices, and a C-terminal helix-turn-helix DNA-binding domain which has been previously observed in the structure the GIY-YIG homing endonuclease I-TevI (van Roey et al. 2001). The ppa-Me motif is composed of two antiparallel P-strands followed by an a-helix and is stabilized by the binding of a metal ion between the first strand and the helix (Kuhlmann et al. 1999).

Although the two families of homing endonucleases share both an active site motif and a DNA-binding domain, their use of these apparently modular structural domains are different. The His-Cys box enzymes have evolved into obligate homodimers whereby two of the P-sheet DNA-binding domains are juxtaposed along a two-fold symmetry axis. This requires pseudopalindromic homing sites, whereas the single copy of this DNA-binding motif in the functional unit of the HNH enzyme allows for more degenerate sequence recognition by removing the palindromic constraint. Furthermore, although HNH endonucleases, His-Cys box endonucleases, and the colicin enzymes all contain the ppa-Me active site motif, their structures indicate that they are used somewhat differently with respect to the chemical identity of metal-binding residues. Specifically, I-Hmul uses both an asparagine and aspartic acid as metal ligands, I-Ppol uses only an asparagine as part of an octahedral magnesium coordination, and the structure of colicin E9 shows a pair of histidine residues binding a metal ion.

The evidence described above supports the hypothesis that HNH and His-Cys box enzymes contain similar DNA-binding motifs and almost identical active sites and therefore are likely to have a common evolutionary ances tor. In fact, the two families are now considered to be divergent members of a single super family of homing endonucleases (see Keeble et al. this Vol).

Acknowledgements. The authors wish to thank P. Haugen for identifying additional His-Cys box endonucleases, and B. Shen and B.L. Stoddard for sharing unpublished results. E.A.G. is supported by a Jane Coffin Childs postdoctoral fellowship.

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