Strukturbasierte Charakterisierung von Klasse II CPD Photolyasen

CPD photolyases are substrate-specific, light-driven DNA repair enzymes which use the energy of sunlight (300 nm – 500 nm) to remove the predominant UV-induced DNA lesion, the cyclobutane pyrimidine dimer (CPD), for ensuring genomic integrity. These monomeric enzymes possess usually two cofactors: the catalytic cofactor FAD crucial for repair activity and an additional antenna chromophore which broadens the photolyase’s action spectrum. Based on sequence identities, CPD photolyases are classified into subtypes with species-specific distributions. While the extensively characterized class I subtype is limited to microbial organisms, the class II subtype is ubiquitous in plants, animals, bacteria, archaea and in some viruses. However, our overall knowledge on the class II photolyases is lagging behind. In this work, functional and structural aspects of the class II subtype were addressed using the archaeal photolyases from the genus Methanosarcina. Due to their high sequence identities to plant and animal orthologs these photolyases are suitable model enzyms to enhance our understanding of class II-specific functionalities. The crystal structures of the Methanosarcina mazei photolyase (MmCPDII) alone and in complex with duplex, CPD-containing DNA provided the first three dimensional information for this phylogenetically distinct group. It became clear that the low discrimination ratio between undamaged and damaged DNA of the class II subtype, which was determined by gel shift experiments, is due to different DNA binding modes. Interestingly, while critical features are functionally similar between the class I and class II subtype, their differences on the structural level suggest that they convergently evolved. For example, during photoreduction of the cofactor FAD the electrons are also transferred along conserved tryptophans, but this pathway takes a different route and the triad of class I is replaced by a dyad with terminal branching points. Comparable to the class I subtype, the neutral semiquinoid species during photoreduction and DNA repair is stabilized by an asparagine. However, in the class II subtype, this conserved residues is located on a different structural element. Finally, use of site-directed mutagenesis and UV/Vis-spectroscopic approaches verified the functional role of these critical features in the class II subtyp. Establishment of an artificial metabolic pathway for providing the deazaflavin 8-HDF in the E. coli expression system finally enabled the identification of the previously unknown antenna chromophore of MmCPDII. The crystal structure of the MmCPDII/8-HDF complex provided detailed insights into the antenna binding pocket and made it possible to draw conclusions regarding potential antennas for the entire class II subtype, especially for the plant photolyases. This information will be a major contribution for the identification of currently cryptic antenna chromophors in plant photolyases.