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In our laboratory, we work for a better understanding of the genotoxic effects of UV radiation and reactive oxygen species (ROS). Our research includes characterization of the damage spectra induced in nuclear and mitochondrial DNA by ROS arising from exogenous (oxidants, photosensitizers, solar radiation) and endogenous (cell metabolism) sources and its contribution to the induction of cancer, ageing, and other diseases. We use a very sensitive alkaline elution method in combination with a panel of specific DNA glycosylases to quantify the generation and repair of oxidative DNA modifications, such as 8-hydroxyguanine in cultured cells and organs of the transgenic mice. We determine the mutagenicity of these lesions in Big Blue mice with and without DNA repair defects and detect the tumor formation in organs. Our group is especially interested in the mechanisms of cellular defense against oxidative DNA damage and its consequences, particularly in the DNA repair mechanisms and their modulation by drugs and endogenous factors.
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The Technique
One focus of our research lies in the interactions of specific DNA lesions with the transcription machineries. A very efficient and versatile DNA repair pathway transcription-coupled nucleotide-excision repair (TCR) is an illustration of enormous importance of such interactions, since it is induced by signals from transcription elongation complexes seized at the DNA adducts. UV radiation is a classical DNA damaging agent that generates pyrimidine dimers that act as transcription blocks. Transcription resumption requires a preceding removal of the lesions by TCR; thus repair can be studied by analyzing the recovery of overall RNA synthesis. However, for the single genes, this assay would be complicated due to the lifetimes of mRNAs and proteins, which are relatively long compared to the repair time. Our approach exploits a green fluorescent protein reporter system wherein messenger RNA and protein sequences are both modified by the attachment of specific degradation signals to achieve sufficiently fast degradation rates. The new reporter system proved useful to detect cessation of transcription after UV-irradiation, as well as resumption of the repaired gene expression.
See “Destabilized green fluorescent protein detects rapid removal of transcription blocks after genotoxic exposure” on page 222.
