The interaction of the EcoRI restriction endodeoxyribonuclease with polynucleotides has been studied in a qualitative manner by an affinity adsorption technique using polynucleotides immobilized on cellulose. It is shown that EcoRI binds to single‐stranded and double‐stranded polyribonucleotides and polydeoxyribonucleotides. Mg2+ ions are not required for binding. In order to define differences between specific and non‐specific binding we have investigated the interaction of EcoRI with d(T‐A‐A‐A‐T‐G), d(T‐T‐A‐C‐A‐T), d(G‐A‐A‐T‐T‐C) and d(G‐G‐A‐A‐T‐T‐C‐C). We have synthesized for this purpose d(T‐A‐A‐A‐T‐G), d(T‐T‐A‐C‐A‐T) and d(G‐A‐A‐T‐T‐C) by the diester approach. d(G‐A‐A‐T‐T‐C) and d(G‐G‐A‐A‐T‐T‐C‐C) are self‐complementary. Differential melting experiments show that the octanucleotide has a melting point of 28°C under ionic conditions where the hexanucleotide is single‐stranded even below 0°C. Correspondingly, the octanucleotide is cleaved by EcoRI, while the hexanucleotide is not. The binding of oligonucleotides to EcoRI can be monitored by the circular dichroism of the enzyme. Titrations show that in the absence of Mg2+ ions all oligonucleotides are bound with similar magnitude: Ka∼ 107 M−1. Complex formation is weakened with increasing temperature, corresponding to a ΔH° of ‐21 kJ/mol and increasing ionic strength, corresponding to an involvement of two ion‐pair bonds between DNA and enzyme. Mg2+ ions have no significant influence on the binding of d(T‐A‐A‐A‐T‐G), d(T‐T‐A‐C‐A‐T) and d(G‐A‐A‐T‐T‐C) to the enzyme. The binding of d(G‐G‐A‐A‐T‐T‐C‐C) to EcoRI is strengthened by a factor of 50 in the presence of Mg2+ ions, as measured by cleavage experiments using d(G‐A‐A‐T‐T‐C) as a competing inhibitor in the enzymatic assay.

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The cleavage of the plasmid pBR322 by the restriction endonuclease EcQ 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 values in the range of 10-4to 10-5 [M nucleotides] . The K. of d-(GGAATTCC3 is 1.5.10-6 [M nucleotides , indicating that the specific binding is approx. 2 orders of magnitude stronger than non-specific binding.