ИСТИНА

For many applications it would be desirable to control DNA cleavage by using an external signal, for example, by light. One of the most often used light-sensitive molecules for regulating functions and properties of nucleic acids and proteins is azobenzene, which can be reversibly isomerized between the extended trans- and the more compact cis-configuration by illumination with light and thus can be used as a "photoswitch". Two novel strategies for regulating the activity of homodimeric proteins, the so called "molecular spring" and "molecular gate" strategy, were proposed and illustrated for the type II restriction endonucleases PvuII (R.PvuII) and SsoII (R.SsoII) as model systems, respectively The "molecular spring" strategy was realized by cross-linking two suitably located cysteine residues of the single chain R.PvuII (scPvuII) with a symmetric azobenzene derivative 4',4-bis(ma1eimido)azobenzene. Under specific illumination the azobenzene moiety containing cross-linker can be isomerized, working like a mechanical spring and changing the local structure of the protein. The DNA cleavage activity of some of the modified R.PvuII variants, which carry the cross-linkers close to the catalytic center, can be modulated by a factor of up to 16 (Schierling et al., 20 10, Proc. Natl. Acad. Sci. USA, 107, 1361 - 1366). The "molecular gate" strategy is based on the fact that most type II restriction endonucleases in the active state are homodimers with the DNA-binding center located in the interface between the two subunits. We have proposed two approaches for this strategy; one making use of a protein modification with azobenzene derivatives that form hydrogen bonds with each other, the other uses a modification that allows forming complexes with a metal ion. These interactions result in a "closed gate" for substrate entry. A two-fold change in the activity of the modified R.SsoII by illumination with UV and blue light, resp. was observed with the two differently modified restriction enzymes. For further developing the "molecular gate" strategy we propose to use azobenzene-containing oligodeoxynucleotides as a steric hindrance for access of the substrate to the DNA-binding center of R.SsoII. It is well known that incorporation of azobenzene moieties into oligodeoxynucleotides dramatically influences the stability of the corresponding duplexes. Formation and dissociation of DNA duplexes can be reversibly photo-regulated by illumination with UV or visible light (Asanuma et al., 2007, Nat. Protoc., 2,203-212). R.SsoII was modified with different self-complementary oligodeoxynucleotides, either containing azobenzene or not, at the entrance of the DNA-binding center. The oligodeoxynucleotides had one or two azobenzene groups. It was shown that the R.SsoII modification with 10- or 14-mer oligodeoxynucleotides prevents enzymatic activity at 25 oC. We have demonstrated that these self-complementary oligodeoxynucleotides form duplexes under these conditions and act as a "closed gate". Upon increasing the temperature (37 oC) and/or illumination with UV light the rates of DNA hydrolysis by R.SsoII increased 3 times, due to destabilization of the duplex and "gate opening".