The Cfr10I restriction enzyme is functional as a tetramer

It is thought that most of TypeII restriction endonucleases interact with DNA as homodimers. The Cfr10I is a typical TypeII restriction enzyme that recognises 5’-PuØCCGGPy sequence and cleaves it as indicated by the arrow. Gel-filtration and analytical ultracentrifugation data presented here indicate that Cfr10I is a homotetramer in solution. The only SfiI restriction enzyme that recognises long interrupted recognition sequence 5’-GGCCNNNNNGGCC has been previously reported to operate as a tetramer, however its structure is unknown. Analysis of Cfr10I crystals revealed that a single molecule in the asymmetric unit is repeated by D2 symmetry to form a tetramer. To determine whether the packing of the Cfr10I in the crystal reflects the quaternary structure of the protein in solution, the tryptophan W220 residue located at the putative dimer-dimer interface was mutated to alanine and the structural and functional consequences of the substitution were analysed. Equilibrium sedimentation experiments revealed that in contrast to the wild type Cfr10I, the W220A mutant exists in solution predominantly as a dimer. In addition, the tetramer seems to be a catalytically important form of Cfr10I since the DNA cleavage activity of the W220A mutant is