The McrBC endonuclease translocates DNA in a reaction dependent on GTP hydrolysis.
McrBC specifically recognizes and cleaves methylated DNA in a reaction dependent on GTP hydrolysis. DNA cleavage requires at least two recognition sites that are optimally separated by 40-80 bp, but can be spaced as far as 3 kb apart. The nature of the communication between two recognition sites was analyzed on DNA substrates containing one or two recognition sites. DNA cleavage of circular DNA required only one methylated recognition site, whereas the linearized form of this substrate was not cleaved. However, the linearized substrate was cleaved if a Lac repressor was bound adjacent to the recognition site. These results suggest a model in which communication between two remote sites is accomplished by DNA translocation rather than looping. A mutant protein with defective GTPase activity cleaved substrates with closely spaced recognition sites, but not substrates where the sites were further apart. This indicates that McrBC translocates DNA in a reaction dependent on GTP hydrolysis. We suggest that DNA cleavage occurs by the encounter of two DNA-translocating McrBC complexes, or can be triggered by non-specific physical obstacles like the Lac repressor bound on the enzyme's path along DNA. Our results indicate that McrBC belongs to the general class of DNA "motor proteins", which use the free energy associated with nucleoside 5'-triphosphate hydrolysis to translocate along DNA.
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Department of Microbiology, Biozentrum, Basel University, Klingelbergstrasse 70, Basel, CH-4056, Switzerland.
Raleigh
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Bickle
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D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport; D004247:DNA / Q000378:metabolism*; D017931:DNA Primers; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D056968:Lac Repressors; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D012097:Repressor Proteins; D006160:Guanosine Triphosphate; D004247:DNA; D004262:DNA Restriction Enzymes; C120301:McrBC endonuclease