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.
Copyright 2000 Academic Press.
MeSH terms
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5-Methylcytosine
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Allosteric Site
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Bacterial Proteins / metabolism
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Base Sequence
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Binding, Competitive
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Coenzymes / metabolism
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Coenzymes / pharmacology
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Cytosine / analogs & derivatives
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Cytosine / metabolism
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DNA Footprinting
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DNA Methylation*
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DNA Restriction Enzymes / metabolism*
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DNA, Bacterial / chemistry*
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DNA, Bacterial / genetics
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DNA, Bacterial / metabolism*
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DNA, Single-Stranded / chemistry
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DNA, Single-Stranded / genetics
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DNA, Single-Stranded / metabolism
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DNA-Binding Proteins / metabolism
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Deoxyribonuclease I / metabolism
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Escherichia coli / enzymology*
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Escherichia coli Proteins*
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Guanosine Triphosphate / metabolism
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Guanosine Triphosphate / pharmacology
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Hydrolysis
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Magnesium / metabolism
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Magnesium / pharmacology
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Models, Biological
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Molecular Sequence Data
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Oligodeoxyribonucleotides / chemistry
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Oligodeoxyribonucleotides / genetics
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Oligodeoxyribonucleotides / metabolism
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Protein Binding / drug effects
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Substrate Specificity
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Thermodynamics
Substances
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Bacterial Proteins
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Coenzymes
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DNA, Bacterial
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DNA, Single-Stranded
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DNA-Binding Proteins
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Escherichia coli Proteins
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Oligodeoxyribonucleotides
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mcrC protein, E coli
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5-Methylcytosine
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Guanosine Triphosphate
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Cytosine
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DNA Restriction Enzymes
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McrBC endonuclease
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mcrB protein, E coli
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Deoxyribonuclease I
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Magnesium