CHARACTERIZATION OF AN EXTREMELY THERMOSTABLE RESTRICTION ENZYME, PSPGI, FROM A PYROCOCCUS STRAIN AND CLONING OF THE PSPGI RESTRICTION-MODIFICATION SYSTEM IN ESCHERICHIA-COLI

An extremely thermostable restriction endonuclease, PspGI, was purifie d from Pyrococcus sp, strain GI-H. PspGI is an isoschizomer of EcoRII and cleaves DNA before the first C in the sequence 5' CCWGG 3' (W is A or T). PspGI digestion can be carried out at 65 to 85 degrees C. To e xpress PspGI at high levels, the PspGI restriction-modification genes (pspGIR and pspGIM) were cloned in Escherichia coli, M.PspGI contains the conserved sequence motifs of alpha-aminomethyltransferases; theref ore, it must be an N4-cytosine methylase, M.PspGI shows 53% similarity to (44% identity with) its isoschizomer, M.MvaI from Micrococcus vari abilis. In a segment of 87 amino acid residues, PspGI shows significan t sequence similarity to EcoRII and to regions of SsoII and StyD4I whi ch have a closely related recognition sequence (5' ?CCNGG 3'), PspGI w as expressed in E. coli via a T7 expression system. Recombinant PspGI was purified to near homogeneity and had a half-life of 2 h at 95 degr ees C, PspGI remained active following 30 cycles of thermocycling; thu s, it can be used in DNA-based diagnostic applications.