Study of the activation mechanism of EcoRII restriction endonuclease using synthetic DNA duplexes

: Efficiency of the cleavage of DNA duplexes with one recognition site by EcoRII restriction endonuclease decreases with the increase in substrate length. DNA duplexes more than 215 base pairs long are practically not cleaved by this enzyme. It has been found that in the presence of substrates 11-14 base pairs long acceleration of hydrolysis of extended single-site substrates by EcoRII enzyme is observed. The level of hydrolysis stimulation is dependent on the length and concentration of the second substrate. A model system for the study of the molecular and kinetic mechanism of EcoRII endonuclease stimulation has been proposed, including a 30-membered single-site substrate and DNA duplexes, modified at heterocyclic bases and internucleotide phosphate groups in the recognition site, as activators. The modified DNA duplexes can activate hydrolysis of the 30-membered substrate and phage T3 DNA. Their influence on the cleavage of extended substrates is dependent on the type of modification and its localization in the recognition site. It has been demonstrated that EcoRII endonuclease carries out cooperative interaction with two recognition sites in DNA.