Interaction of MspI restriction endonuclease with a series of oligodeoxynucleotides, varying in stability of secondary structure and in location of the restriction site, has been studied. It is shown that a functionally active MspI-site must be double-stranded and flanked from both sides. Separate MspI-cleavage of dodecanucleotides dCGACCCGGGATC and dGATCCCGGGTCG is inhibited by the reaction products as well as by non-homological hexanucleotides dGGTACC and dGGATCC (but not by dCGGCGC). Polyethylene glycol in low concentrations (1-3%) promotes and in higher concentrations (7-14%) inhibits the cleavage. A scheme of MspI functioning is suggested including enzyme's step-by-step recognition of the restriction site and its nonspecific interaction with flanking segments of DNA, which leads to formation of the productive complex.
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