Dependence of McrBC cleavage on distance between recognition elements.
DNA cleavage by the modification-dependent restriction enzyme McrBC requires the presence of two suitably modified recognition elements appropriately spaced in the substrate. To characterize the spacing requirement in more detail, we have constructed a plasmid with a single McrBC cleavage site, in which the distance between recognition elements could be systematically varied while preserving the local sequence surrounding the recognition elements. Optimal separation between elements was 55-103 basepairs, with detectable cleavage observed at spacing of 32 bp to 2 kb; no cleavage was seen with spacing of 22 bp or less or with 3 kb between elements. Changing the spacing by 4 basepairs within the optimal range had little effect on the efficiency of cleavage, suggesting that the recognition elements need not lie on the same face of the DNA helix.
Stewart
F J
FJ
New England Biolabs, Beverly, MA 01915, USA.
Raleigh
E A
EA
D001665:Binding Sites; D019175:DNA Methylation; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D029968:Escherichia coli Proteins; D010957:Plasmids / Q000378:metabolism; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D004262:DNA Restriction Enzymes; C120301:McrBC endonuclease; C059678:mcrB protein, E coli