UVR8-based optogenetics and new model systems to study the DNA damage response

This thesis covers the development of a novel genetically encoded light responsive system, or so called optogenetic system, to induce protein-protein interactions, based on the ultraviolet-B sensitive protein UVR8 and its interacting partner COP1 from Arabidopsis thaliana. With such a system, cellular functions can be perturbed instantaneously, while the use of light also enables control over the location, time, and intensity of the perturbation, if suitable optical techniques are available. By making a key step or reaction in a pathway or process light-controlled, optogenetics can be applied to the study of many biological processes, including in the study of DNA damage repair or cell cycle regulation, two processes crucial for maintaining genomic integrity. A specific step is the initial generation of a DNA double strand break. Light controlled dCas9 guided recruitment of endonucleases allows specifying the time in the cell cycle, and the exact location in the genome where a DNA double strand break is produced. This would facilitate the study of repair pathway selection in different cellular contexts. As dCas9 is foreign to human DNA, the consequences of having dCas9 bound to DNA are examined

Ultraviolet-B-mediated induction of protein-protein interactions in mammalian cells

Light-sensitive proteins are useful tools to control protein localization, activation and gene expression, but are currently limited to excitation with red or blue light. Here we report a novel optogenetic system based on the ultraviolet-B-dependent interaction of the Arabidopsis ultraviolet-B photoreceptor UVR8 with COP1 that can be performed in visible light background. We use this system to induce nuclear accumulation of cytoplasmic green fluorescent protein fused to UVR8 in cells expressing nuclear COP1, and to recruit a nucleoplasmic red fluorescent protein fused to COP1 to chromatin in cells expressing UVR8-H2B. We also show that ultraviolet-B-dependent interactions between DNA-binding and transcription activation domains result in a linear induction of gene expression. The UVR8-COP1 interactions in mammalian cells can be induced using subsecond pulses of ultraviolet-B light and last several hours. As UVR8 photoperception is based on intrinsic tryptophan residues, these interactions do not depend on the addition of an exogenous chromophore