Interaction of endonuclease EcoRI with short specific and nonspecific oligonucleotides

The kinetic-thermodynamic analysis of the interaction of endonuclease EcoRI with specific and nonspecific oligonucleotides (ON) of varying structure a nd length showed that any single-stranded ON and their duplexes inhibit the reaction catalyzed by this enzyme. Parameters of affinity of the enzyme to ON and minimum ligands including orthophosphate (K-i = 31 mM) and deoxyrib osophosphate (K-i = 4.6 mM) were determined. The increase in the ON length by one unit up to n less than or equal to 6 enhances their affinity approxi mately twofold. The large number of weak nonspecific contacts of EcoRI with internucleoside phosphates in DNA provides about five orders of magnitude out of the total affinity of ON, whereas the contribution of specific inter actions of EcoRI and DNA does not exceed two orders. The results of analysi s of the EcoRI functioning are in accordance with X-ray analysis data for t he enzyme [1-3]. It remains necessary, however, to specify the concepts of the relative roles of specific and nonspecific DNA-enzyme interactions upon complexation and the necessity of the adjustment of the DNA conformation t o the optimum for the enzyme at the next stage of catalysis to providing sp ecificity of the enzyme action.