SHORT-RANGE AND LONG-RANGE CONTEXT EFFECTS ON COLIPHAGE T4 ENDONUCLEASE II-DEPENDENT RESTRICTION

Synthetic sites inserted into a plasmid were used to analyze the seque nce requirements for in vivo DNA cleavage dependent on bacteriophage T 4 endonuclease II. A 16-bp variable sequence surrounding the cleavage site was sufficient for cleavage, although contest both within and aro und this sequence influenced cleavage efficiency, The most efficiently cleaved sites matched the sequence CGRCCGCNTTGGCNGC, in which the str ongly conserved bases to the left were essential for cleavage. The les s-conserved bases in the center and in the right half determined cleav age efficiency in a manner not directly correlated with the apparent b ase preference at each position; a sequence carrying, in each of the 1 6 positions, the base most preferred in natural sites in pBR322 was cl eaved infrequently. This, along with the effects of substitutions at o ne or two of the less-conserved positions, suggests that several combi nations of bases can fulfill the requirements for recognition of the r ight part of this sequence. The replacements that improve cleavage fre quency are predicted to influence helical twist and roll, suggesting t hat recognition of sequence-dependent DNA structure and recognition of specific bases are both important. Upon introduction of a synthetic s ite, cleavage at natural sites within 800 to 1,500 bp from the synthet ic site was significantly reduced, This suggests that the enzyme may e ngage more DNA than its cleavage site and cleaves the best site within this region, Cleavage frequency at sites which do not conform closely to the consensus is, therefore, highly contest dependent. Models and possible biological implications of these findings are discussed.