Investigation of UVC Induced DNA Damage Formation and Photolyase Catalyzed Repair of Cyclobutane Pyrimidine Dimers

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.