PMID:10788324

Citation	Stewart, FJ, Panne, D, Bickle, TA and Raleigh, EA (2000) Methyl-specific DNA binding by McrBC, a modification-dependent restriction enzyme. J. Mol. Biol. 298:611-22
Abstract	McrBC, a GTP-requiring, modification-dependent endonuclease of Escherichia coli K-12, specifically recognizes DNA sites of the form 5' R(m)C 3'. DNA cleavage normally requires translocation-mediated coordination between two such recognition elements at distinct sites. We have investigated assembly of the cleavage-competent complex with gel-shift and DNase I footprint analysis. In the gel-shift system, McrB(L) binding resulted in a fast-migrating specific shifted band, in a manner requiring both GTP and Mg(2+). The binding was specific for methylated DNA and responded to local sequence changes in the same way that cleavage does. Single-stranded DNA competed for McrB(L)-binding in a modification and sequence-specific fashion. A supershifted species was formed in the presence of McrC and GTPgammaS. DNase I footprint analysis showed modest cooperativity in binding to two sites, and a two-site substrate displayed protection in non-specific spacer DNA in addition to the recognition elements. The addition of McrC did not affect the footprint obtained. We propose that McrC effects a conformational change in the complex rather than a reorganization of the DNA:protein interface.
Links	PubMed Online version:10.1006/jmbi.2000.3697
Keywords	5-Methylcytosine; Allosteric Site; Bacterial Proteins/metabolism; Base Sequence; Binding, Competitive; Coenzymes/metabolism; Coenzymes/pharmacology; Cytosine/analogs & derivatives; Cytosine/metabolism; DNA Footprinting; DNA Methylation; DNA Restriction Enzymes/metabolism; DNA, Bacterial/chemistry; DNA, Bacterial/genetics; DNA, Bacterial/metabolism; DNA, Single-Stranded/chemistry; DNA, Single-Stranded/genetics; DNA, Single-Stranded/metabolism; DNA-Binding Proteins/metabolism; Deoxyribonuclease I/metabolism; Escherichia coli/enzymology; Escherichia coli Proteins; Guanosine Triphosphate/metabolism; Guanosine Triphosphate/pharmacology; Hydrolysis; Magnesium/metabolism; Magnesium/pharmacology; Models, Biological; Molecular Sequence Data; Oligodeoxyribonucleotides/chemistry; Oligodeoxyribonucleotides/genetics; Oligodeoxyribonucleotides/metabolism; Protein Binding/drug effects; Substrate Specificity; Thermodynamics
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