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Kundu, Lal Mohan (2005): Investigation of UVC Induced DNA Damage Formation and Photolyase Catalyzed Repair of Cyclobutane Pyrimidine Dimers. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Gradual depletion of the ozone layer and consequently, increased ultraviolet (UV) radiation on the Earth's surface induces DNA-lesions inside the genome, thereby causing mutations. Three kinds of photoproducts are mainly formed, namely: cyclobutane pyrimidine dimers (CPD), pyrimidine-(6-4)-pyrimidone dimer [(6-4)PP] and the Dewar valence isomer of (6-4)PP lesion. The formation of these photolesions is a major cause of cell death (aging) and fatal disease like skin cancer. A part of this research was performed to investigate the formation and characterization of DNA-lesions under UVC radiation. Small, fluorescent labeled oligonucleotide hairpins (DNA, RNA and mixed DNA-RNA) were employed to this purpose. The amount of damage was quantified using HPLC. Here, a new method was developed, using ion-exchange SAX-column which works at pH = 13, to measure the lesion formation in a direct way. In order to know which lesions are formed, the irradiated hairpins were enzymatically digested. The lesions were separated by HPLC followed by their characterization using MS/MS fragmentation analysis. Investigation was performed to understand the impact of the neighboring nucleobases on the photo-reactivity of a dTpdT-dinucleotide. For this, hairpins were prepared in various sequential contexts. Analysis of these irradiated hairpins revealed the surprising result that the reactivity is strongly reduced when a dTpdT dinucleotide is locked between two neighboring 2'- deoxyguanosines, strongly implying that genomic DNA will be inhomogeneously damaged and hence mutated under UVC radiation. In order to account for the effect of oligonucleotide conformations (A versus B) on the photolesions formation, DNA, RNA and mixed DNA-RNA hairpins were irradiated. The most surprising observation is that the oligonucleotide hairpins, possessing more A-like conformations were found to be very much resistant to UVC degradation. RNA hairpins containing UpU dinucleotides were found to be fully protected from being damaged. A short, dTpdT-containing DNA stretch, embedded in an A-like RNA environment, was also found to be highly stable under UVC light. In the second part of this study, investigation was performed to assay the activities of CPD-photolyase enzyme isolated from different organisms. A synthetic cis-syn thymidine dimer with an open backbone was incorporated into DNA. CPD-photolyases were found to recognize and split this CPDlesion, via a cycloreversion process, into two 2'-deoxythymidine monomers. In order to profile the activities of photolyases and also to study photolyase activity inside living cell, a sensitive DNA-probe, known as molecular beacon was synthesized. The hairpin probe, which features a loop and stem structure contains the model CPD-lesion in its loop region. The molecular beacon (MB 1) in its closed form, is non-fluorescent due to efficient energy transfer (FRET) from the 5'-FAM to 3'-Dabsyl. The MB 1 was designed to undergo strand break on reaction with photolyase in presence of light, thus separating the FAM and the Dabsyl and causing the fluorescence of FAM to be restored. The activities of the photolyase can be studied by monitoring the fluorescence change. Activities of CPDphotolyase isolated from A. nidulans and A. thaliana were studied using this MBprobe. The sensitivity of this probe was tested with wild-type cell-extract from A. thaliana. The fishing-out of photolyase activity from this wild-type extract was possible. Effort was made to investigate the repair process within a living cell, using laser scanning fluorescence microscopy. The insertion of this artificial, chemically modified DNA-substrate in the cell nucleus was achieved.