CHARACTERIZATION OF THE INTERACTION BETWEEN THE RESTRICTION-ENDONUCLEASE MCRBC FROM ESCHERICHIA-COLI AND ITS COFACTOR GTP

McrBC, a GTP-dependent restriction enzyme from E. coli K-12, cleaves D NA containing methylated cytosine residues 40 to 80 residues apart and 3'-adjacent to a purine residue ((PuCN40-80PuC)-C-m-C-m). The presenc e of the three consensus sequences characteristic for guanine nucleoti de binding proteins in one of the two subunits of McrBC suggests that this subunit is responsible for GTP binding and hydrolysis. We show he re that (i) McrB binds GTP with an affinity of 10(6) M-1 and that GTP binding stabilizes McrB against thermal denaturation. (ii) McrB binds GDP about 50-fold and ATP at least three orders of magnitude more weak ly than GTP. (iii) McrB hydrolyzes GTP in the presence of Mg2+ with a steady-state rate of approximately 0.5 min(-1). (iv) McrC stimulates G TP hydrolysis 30-fold, but substrate DNA has no detectable effect on t he GTPase activity of McrB, neither by itself nor in the presence of M crC. (v) Substitution of N339 and N376 with alanine allowed us to iden tify NTAD (339 to 342) rather than NKKA (376 to 379) as the equivalent of the third consensus sequence motif characteristic for guanine nucl eotide binding proteins, NKXD. (C) 1997 Academic Press Limited.