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

The crystal structure of the rare-cutting HNH restriction endonuclease Pad l in complex with its eight-base-pair target recognition sequence 5'-TTAATT AA-3' has been determined to 1.9 angstrom resolution. The enzyme forms an extended homodimer, with each subunit containing two zinc-bound motifs surrounding a beta beta alpha-metal catalytic site. The latter is unusual in that a tyrosine residue likely initiates strand cleavage. Pad l dramatically distorts its target sequence from Watson-Crick duplex DNA base pairing, with every base separated from its original partner. Two bases on each strand are unpaired, four are engaged in noncanonical A:A and T:T base pairs, and the remaining two bases are matched with new Watson-Crick partners. This represents a highly unusual DNA binding mechanism for a restriction endonuclease, and implies that initial recognition of the target site might involve significantly different contacts from those visualized in the DNA-bound cocrystal structures.