Reparatur und Replikation des Sporen Photoproduktes sowie ortsspezifische Generierung eines CPD bzw. Sporen Photoproduktes in DNA

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.