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Methyl-specific DNA binding by McrBC, a modification-dependent restriction enzyme.

F J Stewart·D Panne·T A Bickle·E A Raleigh
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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.

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5-MethylcytosineAllosteric SiteBacterial ProteinsBase SequenceBinding, CompetitiveCoenzymesCytosineDNA FootprintingDNA MethylationDNA Restriction EnzymesDNA, BacterialDNA, Single-StrandedDNA-Binding ProteinsDeoxyribonuclease IEscherichia coliEscherichia coli ProteinsGuanosine TriphosphateHydrolysisMagnesiumModels, BiologicalMolecular Sequence DataOligodeoxyribonucleotidesProtein BindingSubstrate SpecificityThermodynamicsJournal Article

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F J Stewart, D Panne, T A Bickle, E A Raleigh. 2000-05-12. Methyl-specific DNA binding by McrBC, a modification-dependent restriction enzyme.. https://doi.org/10.1006/jmbi.2000.3697

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