Methyl-specific DNA binding by McrBC, a modification-dependent restriction enzyme.
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.
Stewart
F J
FJ
New England Biolabs, Inc, Beverly, MA 01915, USA.
Panne
D
D
Bickle
T A
TA
Raleigh
E A
EA
D044503:5-Methylcytosine; D000495:Allosteric Site; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001667:Binding, Competitive; D003067:Coenzymes / Q000378:metabolism / Q000494:pharmacology; D003596:Cytosine / Q000031:analogs & derivatives / Q000378:metabolism; D018983:DNA Footprinting; D019175:DNA Methylation; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D006868:Hydrolysis; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008954:Models, Biological; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D013379:Substrate Specificity; D013816:Thermodynamics
D001426:Bacterial Proteins; D003067:Coenzymes; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; C069326:mcrC protein, E coli; D044503:5-Methylcytosine; D006160:Guanosine Triphosphate; D003596:Cytosine; D004262:DNA Restriction Enzymes; C120301:McrBC endonuclease; C059678:mcrB protein, E coli; D003850:Deoxyribonuclease I; D008274:Magnesium