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, 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.
Gast
F U
FU
Institut für Biochemie, Justus-Liebig-Universität Giessen, Germany.
Brinkmann
T
T
Pieper
U
U
Krüger
T
T
Noyer-Weidner
M
M
Pingoud
A
A
D001482:Base Composition; D001665:Binding Sites / Q000187:drug effects; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects; D004262:DNA Restriction Enzymes / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D006160:Guanosine Triphosphate / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D006868:Hydrolysis; D016296:Mutagenesis / Q000235:genetics
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D006160:Guanosine Triphosphate; D004247:DNA; D004262:DNA Restriction Enzymes; C059678:mcrB protein, E coli