THE RECOGNITION OF METHYLATED DNA BY THE GTP-DEPENDENT RESTRICTION-ENDONUCLEASE MCRBC RESIDES IN THE N-TERMINAL DOMAIN OF MCRB

McrBC is a GTP-dependent restriction endonuclease of E, coli K12, sele ctively directed against DNA containing modified cytosine residues, Mc rB, one of its components, is responsible for the binding and, togethe r with McrC, for the cleavage of DNAs containing two 5'-(PuC)-C-m site s separated by 40-80 base pairs. Gel retardation assays with wild-type and mutant McrB reveal that (i) single 5'-(PuC)-C-m sites in DNA can be sufficient to elicite binding by McrB, Binding to such substrates i s, however, weak and strongly dependent on the sequence context of (Pu C)-C-m sites, (ii) Strong DNA binding (K-ass similar to 10(7) M-1) is dependent on the presence of at least two (PuC)-C-m sites, even if the y are separated by less than 40 bp, and is modulated by the sequence c ontext CGGT->-A(m)CT(C)/(G)AGT->-AGG(m)CCT->-AAG(m)CTT-), (iii) DNA bi nding by McrB is accompanied by formation of distinct multiple complex es whose distribution is modulated by GTP, (iv) McrC, which cannot bin d DNA by itself, moderately stimulates the DNA binding of McrB and con verts McrB-DNA complexes to large aggregates, (v) Deletion of the C-te rminal half of McrB, which harbors the three consensus sequences chara cteristic for guanine nucleotide binding proteins, leads to protein in active in GTP binding and/or hydrolysis and in McrC-assisted DNA cleav age; the protein, however, remains fully competent in DNA binding, (vi ) Mutations in McrB which read to a reduction in GTP binding and/or hy drolysis can affect DNA binding, suggesting that the two activities ar e coupled in the full-length protein.