The interaction of MvaI restriction endonuclease with 14-membered deoxyribonucleotide duplexes containing modifications within the recognition site (CCA/TGG) has been studied. Substitution of m'dC for the internal dC residue, as well as substitution of fl'dU or rU for dT did not influence the initial rate of hydrolysis (vo) of modified strands, whereas the hydrolysis of unmodified strands was inhibited in some cases. Furthermore, the substitution of a pyrophosphate bond for a scissile phosphodiester bond in one strand completely inhibited digestion in this strand without any decrease of the rate of hydrolysis of the unmodified strand. In contrast to EcoRII endonuclease, which recognizes the same DNA sequence, in the case of MvaI endonuclease substrate recognition is possible in a wide range of conformational, electronic and hydrophobic alterations within the recognition site.

A new photo-cross-linking dU analog, 5-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl]-2'-deoxyuridine, was synthesized and incorporated into the recognition site of EcoRII and MvuI restriction-modification enzymes. The resulting base-modified 14-mer substrate was tested for cross-linking to these enzymes. Cross-linking is effected by irradiation of the enzyme-substrate complexes at 366 nm.

The efficiency of cleavage of DNA duplexes with single EcoRII recognition sites by the EcoRII restriction endonuclease decreases with increasing substrate length. DNA duplexes of more than 21 5 bp are not effectively cleaved by this enzyme. Acceleration of the hydrolysis of long single-site substrates by EcoRII is observed in the presence of 11 -14-bp substrates. The stimulation of hydrolysis depends on the length and concentration of the second substrate. To study the mechanism of EcoRII endonuclease stimulation, DNA duplexes with base analogs and modified internucleotide phosphate groups in the EcoRII site have been investigated as activators. These modified duplexes are cleaved by EcoRII enzyme with different effciences or are not cleaved at all. It has been discovered that the resistance of some of them can be overcome by incubation with a susceptible canonical substrate. The acceleration of cleavage of long single-site substrates depends on the type of modification of the activator. The modified DNA duplexes can activate EcoRII catalyzed hydrolysis if they can be cleaved by EcoRII themselves or in the presence of the second canonical substrate. It has been demonstrated that EcoRII endonuclease interacts in a cooperative way with two recognition sites in DNA. The cleavage of one of the recognition sites depends on the cleavage of the other. We suggest that the activator is not an allosteric effector but acts as a second substrate.EcoRII restriction endonuclease recognizes the (5')JCCiGG (3') sequence in DNA and cleaves it prior to the first nucleotide [l].It has been found recently [2, 31 that the mechanism of EcoRIIIDNA interaction is more complex than that of type-I1 restriction enzymes studied before [4, 51. This enzyme is not able to cleave phage T3 DNA with three EcoRII sites and phage T7 DNA with the unique EcoRII site (the single-site effect). Addition of susceptible DNA or short substrates may, however, stimulate such a cleavage. It was suggested that EcoRII requires at least two recognition sites for its activation [3]. In order to substantiate the mechanism of EcoRIIlsubstrate interaction, we describe here a useful model system to study the stimulation effect and to test a wide set of DNA duplexes containing modified and unmodified EcoRII sites as activators. The main purpose of using differently modified oligonucleotides was to determine the requirements for the activator: its structure and ability to be cleaved by EcoRII. Substrate analogs were chosen according to our recent findings that they are cleaved by EcoRII with different efCorrespondence to E. S. Gromova, A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow W-234, Russia, 119899Ahbveviations. m4C, N4-methyldeoxycytidine; m6A, N6-methyldeoxyadenosine; flsU, 5-fluorodeoxyuridine.Enzymes. Restriction endonucleases EcoRII and MvaI (EC 3.1.21.4); T4polynucleotide kinase (EC 2.7.1.78); T4 DNA ligase (EC 6.5.1.1). ficiencies or are not cleaved at all [6, 71. Additionally, the length of the substrates producing...