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McrBC is a GTP-dependent restriction endonuclease of E. coli K12, selectively directed against DNA containing modified cytosine residues. McrB, one of its components, is responsible for the binding and, together with McrC, for the cleavage of DNAs containing two 5'-Pu(m)C sites separated by 40-80 base pairs. Gel retardation assays with wild-type and mutant McrB reveal that (i) single 5'-Pu(m)C sites in DNA can be sufficient to elicite binding by McrB. Binding to such substrates is, however, weak and strongly dependent on the sequence context of Pu(m)C sites. (ii) Strong DNA binding (K(ass) approximately 10(7)M[-1]) is dependent on the presence of at least two Pu(m)C sites, even if they are separated by less than 40 bp, and is modulated by the sequence context (-A(m)CCGGT- --> -A(m)CT(C/G)AGT- --> -AGG(m)CCT- --> -AAG(m)CTT-). (iii) DNA binding by McrB is accompanied by formation of distinct multiple complexes whose distribution is modulated by GTP. (iv) McrC, which cannot bind DNA by itself, moderately stimulates the DNA binding of McrB and converts McrB-DNA complexes to large aggregates. (v) Deletion of the C-terminal half of McrB, which harbors the three consensus sequences characteristic for guanine nucleotide binding proteins, leads to protein inactive in GTP binding and/or hydrolysis and in McrC-assisted DNA cleavage; the protein, however, remains fully competent in DNA binding. (vi) Mutations in McrB which lead to a reduction in GTP binding and/or hydrolysis can affect DNA binding, suggesting that the two activities are coupled in the full-length protein.

Restriction of DNA by the Escherichia coli K‐12 McrBC restriction endonuclease, which consists of the two subunits McrB and McrC, depends on the presence of modified cytosine residues in a special constellation. From previous work by others it was known that restriction of 5‐methylcytosine‐containing DNA requires two methylated 5′‐PuC sites separated by approximately 40‐80 non‐defined base pairs. Here we show that binding of the McrBC nuclease is mediated exclusively by the McrB subunit. McrB has a low affinity for non‐methylated DNA, with which it forms low molecular weight complexes. The affinity for DNA is significantly increased, with variations depending on the sequence context, by hemi‐ or fully methylated 5′‐PuC sites. Binding to such substrates yields high molecular weight complexes, presumably involving several McrB molecules. Methylation at unique 5′‐PuC sites can be sufficient to stimulate DNA binding by McrB. As such substrates are not cleaved by the nuclease, restriction apparently requires the coordinated interaction of molecules bound to neighbouring 5′‐PumC sites. The binding properties of McrB exhibit some similarities to recently identified eukaryotic proteins interacting in a non‐sequence‐specific manner with DNA containing methylated 5′‐CpG sequences and might point to a common molecular origin of these proteins. In addition to DNA, McrB also binds GTP, an essential cofactor in DNA restriction by McrBC. McrC neither binds to DNA nor modulates the DNA binding potential of McrB. As McrC is essential for restriction it appears to predominantly function in catalysis.

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