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The three-dimensional crystal structures of HindIII bound to its cognate DNA with and without divalent cations were solved at 2.17 and 2.00 Å resolution, respectively. HindIII forms a dimer. The structures showed that HindIII belongs to the EcoRI-like (α-class) subfamily of type II restriction endonucleases. The cognate DNA-complex structures revealed the specific DNA-recognition mechanism of HindIII by which it recognizes the palindromic sequence A/AGCTT. In the Mg2+ ion-soaked structure the DNA was cleaved and two ions were bound at each active site, corresponding to the two-metal-ion mechanism.