title: Controlling the DNA cleavage activity of light-inducible chimeric endonucleases by bidirectional photoactivation.
abstract: A functional coupling of photosensory domains derived from photoreceptors to effector proteins is a promising strategy for engineering novel photoresponsive proteins in optogenetics. Here, we have fused the light-sensitive LOV2 domain from Avena sativa phototropin1 to the restriction enzyme PvuII to generate a genetically encoded, light-controllable endonuclease. By analyzing several LOV-PvuII fusion enzymes, variants were obtained that show a 3-fold difference in DNA cleavage activity, when illuminated with blue light or kept in the dark. The effect is fully reversible over multiple photocycles. Depending on the particular fusion interface, the LOV-PvuII variants obtained had a bidirectional polarity in photoactivation; i.e., increased DNA cleavage activity was observed either in the dark state, with a compact folded LOV domain, or in the blue light photoexcitation state, when the LOV domain is partially unfolded.
authors.affiliation: Institute of Biochemistry, Justus-Liebig University , Heinrich-Buff-Ring 58, D-35392 Giessen, Germany.
authors.forename: Benno
authors.initials: B
authors.lastname: Schierling
authors.forename: Alfred
authors.initials: A
authors.lastname: Pingoud
mesh_terms: D000595:Amino Acid Sequence; D018554:Avena / Q000201:enzymology / Q000235:genetics*; D001665:Binding Sites; D053837:DNA Cleavage; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008027:Light; D008958:Models, Molecular; D008969:Molecular Sequence Data; D055668:Photochemical Processes; D056949:Phototropins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D056949:Phototropins; D011993:Recombinant Fusion Proteins; C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific