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Mees, Alexandra (2005): Co-Kristallisation von DNA-Photolyase aus A. nidulans mit Thymidindimer enthaltender DNA: Einbau von Flavin- und Desazaflavin-Cofaktoren in Oligonukleotide. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

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.