The Cfr10I restriction enzyme is functional as a tetramer

It is thought that most of the type II restriction endonucleases interact w ith DNA as homodimers. Cfr10I is a typical type II restriction enzyme that recognises the 5 '-Pu(down arrow)CCGGPy sequence and cleaves it as indicate d by the arrow. Gel-filtration and analytical ultracentrifugation data pres ented here indicate that Cfr10I is a homotetramer in isolation. The only Sf iI restriction enzyme that recognises the long interrupted recognition sequ ence 5 '-GGCCNNNNNGGCC has been previously reported to operate as a tetrame r however, its structure is unknown. Analysis of Cfr10I crystals revealed t hat 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 crys tal reflects the quaternary structure of the protein in solution, the trypt ophan W220 residue located at the putative dimer-dimer interface was mutate d to alanine, and the structural and functional consequences of the substit ution were analysed. Equilibrium sedimentation experiments revealed that, i n contrast to the wildtype Cfr10I, the W220A mutant exists in solution pred ominantly as a dimer. In addition, the tetramer seems to be a catalytically important form of Cfr10I, since the DNA cleavage activity of the W220A mut ant is <0.1% of that of the wild-type enzyme. Further, analysis of plasmid DNA cleavage suggests that the Cfr10I tetramer is able to interact with two copies of the recognition sequence, located on the same DNA molecule. Inde ed, electron microscopy studies demonstrated that two distant recognition s ites are brought together through the DNA looping induced by the simultaneo us binding of the Cfr10I tetramer to both sites. These data are consistent with the tetramer being a functionally important form of Cfr10I. (C) 1999 A cademic Press.