Inhibition of Eco RI action by polynucleotides. A characterization of the non-specific binding of the enzyme to DNA

Abstract

The cleavage of the plasmid pBR322 by the restriction endonuclease Eco RI has been studied in the presence of various polynucleotides and the double stranded octanucleotide d-(GGAATTCC) in order to clarify whether there is a preferential interaction of Eco RI with DNA sequences other than -GAATTC-. The steady state kinetic analysis shows that all polynucleotides investigated with the possible exception of poly-dG·poly-dC inhibit the cleavage competitively with K _j values in the range of 10 ⁻⁴ 10 ⁻⁵ [M nucleotides] The K _j of d-(GGAATTCC is 1.5·10 ⁻⁶ [M nucleotides], indicating that the specific binding is approx. 2 orders of magnitude stronger than non-specific binding.