Strukturelle Aufklärung und funktionelle Charakterisierung der DNA-Reparaturprozesse in DNA-Photolyasen und Cryptochrom 3

Die Einwirkung von kurzwelliger UV-Strahlung führt in allen Organismen durch die Bildung von Photoaddukten zu schwerwiegenden Schädigungen der die Erbinformationen tragenden DNA. Die überwiegend auftretenden Photoschäden sind hierbei Cyclobutanpyrimidindimere (CPDs), die durch eine photochemisch ind...

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Bibliographic Details
Main Author: Klar, Tobias
Contributors: Essen, Lars-Oliver (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2007
Online Access:PDF Full Text
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The influence of short wave ultraviolet radiation can cause severe DNA damages by forming photoadducts. The major UV-induced photolesions are cyclobutane-pyrimidine dimers (CPDs) which are formed in a photochemically induced cyclization reaction of two adjacently located pyrimidine bases, mainly thymine. To ensure the genetic integrity many organisms contain DNA photolyases. These enzymes are capable of repairing DNA lesions by blue light induced cleavage of the dimer. The full details of the repair mechanism are not yet clear. Only three protein structures of photolyases without any detail on substrate binding were available. One of the aims of this study was to elucitate the binding and repair mechanism of photolyases by cocrystallization of a photolyase and CPD-comprising DNA. This study presents the 1.8 Å cocrystal structure of A. nidulans photolyase bound to duplex DNA comprising a synthetic CPD lesion. The structure reveals the CPD lesion completely turned out of the double helix and flipped into the active site. Interestingly, the thymine dimer was found to be split into two thymines by synchrotron radiation at 100 K. The structure mimics a structural intermediate during DNA repair in which back-flipping of the thymines into duplex DNA has not yet taken place. Both, the dinucletide flipping mechanism which has been a long-standing hypothesis, supported by many computer model calculations, biochemical data and NMR spectroscopy, and the DNA bending of 50 ° upon DNA binding could be confirmed with this cocrystal structure. Likewise, the exact geometry of the catalytic flavin cofactor and the precise binding mode between the CPD lesion and the photolyase could be elucidated. Observed interactions between DNA and the photolyase define a 5-NpT◊TpNpNp-3 signature as a common signature motif of DNA binding by photolyases. Structural considerations give rise to either a direct tunneling electron transport pathway from the C8 methyl group of the FAD cofactor to the CPD lesion or an indirect pathway involving the adenine moiety. The structural insights gained from this work provide valuable information on the detailed understanding of the most important direct DNA repair process. Cryptochromes are closely related to photolyases and are part of the same superfamily. As a member of the blue light photoreceptor family they are known to regulate the circadian rhythm in numerous organisms. The second part of this thesis deals with the structure determination and characterization of A. thaliana cryptochrome 3 (cry3) which belongs to the class of the so called DASH-crpytochromes. Up to know, the physiological function of this class of cryptochromes is mostly unknown. The crystal structure of A. thaliana cry3 was solved at a resolution of 1.9 Å, revealing MTHF as an antenna cofactor. Furthermore, the cocrystal structure of A. thaliana cry3 bound to a pentanucleotide comprising a CPD lesion could be obtained and successfully characterized by X-ray crystallography at a resolution of 2.0 Å. This structure reveals the DNA lesion bound in its repaired state before back flipping of the two thymines out of the active site, in analogy to the cocrystal structure with the A. nidulans photolyase decribed above. Additionally, the observed binding mode of DNA to A. thaliana cry3 mainly corresponds to that monitored in photolyases leading to the assumption that the electron transfer pathways should be mostly identical. Photolyases contain either 8-HDF, MTHF or FAD as an antenna cofactor to broaden their activity spectrum. The ELISA activity assays on T. thermophilus photolyase carried out in the third part of this thesis show an eightfold higher repair activity at 450 nm for the FMN-containing photolyase compared to the activity of the cofactor-free R46E mutant of the enzyme. Thus, the importance of FMN as an alternative antenna chromophore could be confirmed. Reconstitutions of the native photolyase with the naturally occuring cofactors FMN and 8-HDF as well as the artificial 8-iodo-8-demethyl-riboflavin (8-IRF) and subsequent crystallographic analysis of the binding modes clearly showed the promiscuity of the antenna pigment binding site. With the insertion of various synthesized antenna chromophores possessing different spectral properties it should now be possible to broaden the activity spectrum for photolyase DNA repair.