Controlling the DNA cleavage activity of light-inducible chimeric endonucleases by bidirectional photoactivation

Bioconjugate Chemistry
Benno Schierling, Alfred Pingoud

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.

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Citations

Feb 16, 2013·Molecular BioSystems·Konrad Müller, Wilfried Weber
Oct 2, 2013·Planta·Arash Kianianmomeni, Armin Hallmann
Nov 19, 2014·BMC Systems Biology·Svetlana UsherenkoChristof Taxis
Feb 16, 2016·Microbial Cell Factories·Mitsuharu NakajimaKoji Sode
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Dec 12, 2012·Molecular BioSystems·Ishtiaq Ahmed, Ljiljana Fruk
May 12, 2019·Nature Protocols·Onur DagliyanKlaus M Hahn
Feb 18, 2017·Applied Microbiology and Biotechnology·Francisco SalinasLuis F Larrondo

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