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Pieck, Jan Carsten (2007): Reparatur und Replikation des Sporen Photoproduktes sowie ortsspezifische Generierung eines CPD bzw. Sporen Photoproduktes in DNA. Dissertation, LMU München: Fakultät für Chemie und Pharmazie



Damaged DNA leads to biological processes such as mutagenesis and apoptosis. Deeper knowledge about DNA lesions, DNA lesion recognition and lesion repair or error-free DNA replication is essential to understand and to prevent these processes. This thesis describes investigations of the UV-light inducible spore photoproduct (SP) and the cyclobutane-pyrimidine-dimer (CPD). In the first part of this work the preparation and purification of a single stranded DNA containing a site-specific defined spore photoproduct is described. This DNA lesion is inducible upon UV-light irradiation of DNA in the presence of pyridinedicarboxylic acid. This SP lesion is the only detectable UV light inducible DNA lesion in the spores of Bacillus and Clostridium species. The limitation to only one UV light lesion is one of the major reasons for the UV resistance of spores, since only one enzyme is necessary for the lesion repair. This repair enzyme is called spore photoproduct lyase. For enzymatic repair studies of the spore photoproduct, the first thermophilic spore photoproduct lyase of G. stearothermophilus (SplG) was cloned, over-expressed, purified and successfully reconstituted. The single stranded DNA containing a spore photoproduct lesion is efficiently repaired by the spore photoproduct lyase. The catalytic activity is 2,6 µmol sporephotoproduct repaired per minute and mg SplG, which results in a calculated turnover of ~100. In the second part of this work, the in vivo and in vitro replication of the spore photoproduct was investigated. For the in vitro assays one high fidelity polymerase of the family A, BstPol1, and two members of the low fidelity family Y, DinB and pol η, were used.