DEPENDENCE OF MCRBC CLEAVAGE ON DISTANCE BETWEEN RECOGNITION ELEMENTS

DNA cleavage by the modification-dependent restriction enzyme McrBC re quires the presence of two suitably modified recognition elements appr opriately spaced in the substrate. To characterize the spacing require ment in more detail, we have constructed a plasmid with a single McrBC cleavage site, in which the distance between recognition elements cou ld be systematically varied while preserving the local sequence surrou nding the recognition elements. Optimal separation between elements wa s 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 wit h 3 kb between elements. Changing the spacing by 4 basepairs within th e optimal range had little effect on the efficiency of cleavage, sugge sting that the recognition elements need not lie on the same face of t he DNA helix.