Crystallization and preliminary X-ray analysis of restriction endonuclease FokI bound to DNA

FokI is a type Ils restriction endonuclease which recognizes an asymmetric DNA sequence and cleaves DNA a short distance away from the sequence, The enzyme is bipartite in nature with its DNA recognition and cleavage functions located on distinct domains, We report here cocrystals of the complete FokI enzyme (579 amino acids) hound to a 20-bp DNA fragment containing its recognition sequence, The complex is amongst the largest protein-DNA complexes to he crystallized, and required macroseeding techniques for optimal crystal growth, The cocrystals diffract to at least 2.8 Angstrom in resolution and belong to space group P2(1) with unit cell dimensions of a = 67.9 Angstrom, b = 119.8 Angstrom, c = 69.1 Angstrom, beta = 96.6 degrees. Using specific amino acid analysis we show that asymmetric unit contains a single FokI molecule bound to the 20-bp DNA fragment, This paper reports the first cocrystals of a type IIs restriction endonuclease. (C) 1997 Federation of European Biochemical Societies.