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Kinetic properties of optogenetic site-specific DNA recombination by LiCre-loxP.
Biol Open, 15(5).
Advances in optogenetics now allow specific modifications to the DNA of livecells with light. However, successfully using these technologies requires knowingtheir properties in terms of sensitivity, efficiency, kinetics and mechanism. Wepreviously developed an optogenetic tool made of a single chimeric protein calledLiCre that enables the induction of specific changes in the genome with bluelight via DNA recombination between loxP sites ( Duplus-Bottin et al., 2021).Here, we used in vitro and in vivo experiments combined with kinetic modeling toprovide a deeper characterization of the photoactivated LiCre-loxP recombinationreaction. We find that LiCre binds DNA with high affinity in the absence of alight stimulus and that this binding is cooperative, although not as much as forthe Cre recombinase from which LiCre was derived. In yeast, the addition ofriboflavin to the culture medium had no effect on LiCre's efficiency, even whencells over-expressed riboflavin kinase, suggesting that the abundance of theflavin mononucleotide cofactor is not limiting for the reaction. However, LiCre'sefficiency in yeast gradually increased when raising the temperature from 20°C to37°C. The recombination kinetics observed in live cells are best explained by amodel where the photoactivation of two or more DNA-bound LiCre units (happeningin seconds) can produce (in several minutes) a functional recombination synapse.This model was able to capture the effect of a point mutation altering LiCre'slight cycle. This deeper understanding of the LiCre-loxP system providesadditional knowledge for designing experiments where specific genetic changes areinduced in live cells with light.
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