Abstract
Type I restriction-modification systems bind to non-palindromic, bipartite recognition sequences. Although these enzymes methylate specific adenine residues within their recognition sequences, they cut DNA at sites up to several thousand base-pairs away. We have investigated the mechanism of how EcoR124II, a type IC restriction-modification system, selects the cleavage site. Restriction studies with different DNA constructs revealed that circular DNA requires only one non-methylated recognition sequence to be cut, whereas linear DNA needs at least two such sites. Cleavage of linear DNA is independent of site orientation. Further investigations of the linear substrates revealed a mechanism whereby the double-strand break is introduced between two recognition sequences. We propose a model for the selection of restriction sites by type I enzymes where two EcoR124II complexes bind to two recognition sequences. Lack of methylation at a site stimulates the enzyme to translocate DNA on both sides of the recognition sequence. Thus the two complexes approach each other and, at the point where they meet, they interact to introduce a double-strand break in the DNA.
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