Co-Kristallisation von DNA-Photolyase aus A. nidulans mit Thymidindimer enthaltender DNA: Einbau von Flavin- und Desazaflavin-Cofaktoren in Oligonukleotide

Since the beginning of life on earth, cells and their DNA have been
exposed to sunlight. Short-wavelength UV-irradiation causes severe
genomic damage as a result of the formation of photoproducts in
DNA. The major UV-induced DNA lesion is the cis,syn cyclobutane
pyrimidine dimer (CPD) which is formed between two adjacent
pyrimidine bases, normally thymidines. These photolesions, if left
unrepaired, can be mutagenic and carcinogenic. For this reason it
is currently of great interest to learn about the detection and
repair of UV-induced DNA lesions. In prokaryotes, plants and some
vertebrates DNA photolyases are responsible for the repair of these
photolesions. Photolyases are coenzyme-dependent repair enzymes
which cleave the dimer into the monomers in a sunlight-initiated
process thereby regenerating the intact DNA. Although three crystal
structures of photolyases have been solved, it has been difficult
to obtain a co-crystal structure of this enzyme together with the
substrate: DNA containing CPD lesions. The structure of the
photolyase/substrate complex is of great importance, as this
facilitates the complete elucidation of the binding and repair
mechanisms of the enzyme. Photolyases of the deazaflavin-class
contain as a light harvesting cofactor 8-hydroxy-5-deazaflavin, the
F0 cofactor. Since the F0 cofactor cannot be synthesized during the
overexpression process of A. nidulans photolyase in E. coli, the F0
was synthesized chemically as a part of this work and then
incorporated into the enzyme. For the DNA substrate, a thymidine
dimer lesion with a formacetal bridge was incorporated into
oligonucleotides by solid phase synthesis and then crystallised
with the protein. In this Ph. D. thesis a co-crystal structure of
A. nidulans photolyase in complex with CPD-containing duplex DNA
was successfully elucidated. The 1.8 Å crystal structure shows the
dimer lesion being completely flipped out of the duplex DNA into
the active site of the enzyme and split into the two corresponding
thymines by synchrotron radiation at 100 K. This process is
accompanied by additional bending of the DNA to about 50 °. The
structure apparently mimics a structural sub-state during
light-driven DNA repair in which back flipping of the thymines into
duplex DNA has not yet taken place. The co-crystal structure
described in this thesis represents the first structure which can
confirm the hitherto only postulated dinucleotide flipping
mechanism of photolyases. Photolyases of the deazaflavin class
possess as cofactors one flavin and one deazaflavin moiety. Due to
their interesting chemical and physical properties even these
cofactors themselves can form the basis of variable and fundamental
investigations. In nature flavin-dependent enzymes play a crucial
role in the catalysis of redox reactions. Due to growing interest
in biocatalysis based on oligonucleotide structures, the
incorporation of coenzymes into DNA or RNA is of high interest. The
construction of flavin-dependent ribozymes with properties that
extend into the domain of redox catalysis requires detailed
knowledge of the fluorescence properties of coenzymes in an
oligonucleotide environment. A riboflavin cofactor was incorporated
into DNA which could potentially replace the protein environment of
coenzyme-dependent enzymes. This thesis presents the first study of
the complex fluorescence behaviour of an artificial flavin base in
DNA. The coenzyme is surprisingly useful for the precise monitoring
of nucleobases in its vicinity. The described results will be the
basis for further investigations into flavin-containing
bioanalytical devices. The deazaflavin cofactor is another very
common coenzyme with differing functions. It acts as redox cofactor
F420 in methanogenic bacteria and as F0 it is a light harvesting
photoantenna in many photolyases. In this work a deazaflavin
building block was synthesized which was incorporated into DNA. For
the first time a deazaflavin has been incorporated within an
oligonucleotide via phosphodiester bonds. Together with an embedded
flavin cofactor and pyrimidine lesions, a new complete model system
of photolyase with DNA as a fixed pre-organising template can be
generated. This will allow systematic investigations into the
distance dependence of energy transfer processes between
deazaflavin and flavin, and into repair processes. The results from
this thesis yielded fascinating insights into one of the most
important genome repair processes in nature.