EcoRII Restriction Endonuclease Forms Specific Contacts to the Bases of Its Target Sequence Flipped from DNA in a Transition Complex with Photoactivatable Substrates

The photoactivatable modified oligonucleotides were used to investigate direct contacts formed by the type IIE EcoRII restriction endonuclease and T/A bases of its recognition site (5'-CCT/AGG). dimer consists a central catalytic core, made two C-terminal endonuclease-like domains (EcoRII-C) from different subunits, N-terminal effector DNA binding (EcoRII-N). According co-crystal structure isolated EcoRII-C with flips nucleotides pair into enzyme pockets. Нere, photocross-linking technique was extrahelical in protein pockets full-length within pre-reactive EcoRII–DNA complex obtained presence Ca2+ solution. Photoreactive zero-length agent 5-iodo-2'-deoxyuridine (IdU) introduced as single substituent position or flanking nucleotide sequences 14-mer substrate. substitution only dT dA residues resulted formation photocross-links upon irradiation Ca2+. Proteolytic digestion enzyme-oligonucleotide conjugates followed MALDI-MS analysis have allowed identify 224VEYD227 region involved cross-links. This belongs part H-10 α-helix. Y226 residue suggested form cross-link T A replaced IdU complex. flipped base seem accommodate equally well both Altogether, IdU-containing substrates trap before cleavage solution enzyme–DNA important for high specificity Т/A providing highly specific reaction.