Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 298, Issue 4, Pages 611-622Publisher
ACADEMIC PRESS LTD
DOI: 10.1006/jmbi.2000.3697
Keywords
nucleotide-dependent; GTP-dependent; gel-shift; footprint; Rg1B
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McrBC, a GTP-requiring, modification-dependent endonuclease of Escherichia coli K-12, specifically recognizes DNA sites of the form 5' (RC)-C-m 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 Mg2+. 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 GTP gamma S. 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. (C) 2000 Academic Press.
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