SYNTHETIC DNA DUPLEXES AS A TOOL IN STUDYING THE MECHANISM OF ECORII ACTIVATION

The efficiency of EcoRII cleavage of synthetic DNA duplexes with one E coRII recognition site decreases with increasing substrate length. Thi s enzyme virtually fails to cleave DNA duplexes longer than 215 bp. Ho wever, EcoRII cleaves long DNA duplexes with one recognition site in t he presence of 11-14-bp substrates. The extent of hydrolysis activatio n depends on the length and concentration of the added substrate. A mo del system is suggested for studying the molecular and kinetic mechani sm of EcoRII activation. This system includes a 30-bp substrate with o ne EcoRII recognition site, and DNA duplexes as activating substrates that contain modified heterocyclic bases and internucleotide phosphate groups. DNA duplexes with a modified EcoRII recognition site may acti vate hydrolysis of the 30-bp substrate and of phage T3 DNA. Their cata lytic effect on the cleavage of extended duplexes depends on the type of modification and its localization in the recognition site. Cooperat ive interaction of EcoRII with two recognition sites in DNA has been s hown to be essential for the functioning of the enzyme.