Structure and dynamics of novel bacterial photolyases

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Photolyases and cryptochromes form a superfamily (PCSf) of light-driven proteins occurring in all domains of Life. CPD and (6-4) photolyases repair UV induced DNA lesions, cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts (6-4PP), after photoexcitation of their fully reduced flavin adenine dinucleotide cofactor, FADH(-). Cryptochromes confer many biological light responses and depend for that on the flavin photoreduction processes known from photolyases. Most photolyases and cryptochromes have intimate evolutionary relationships by conserving major structural and functional features of their photoactive regions. In contrast, some PCSf families are only distantly related to these canonical and well-studied photolyases/cryptochromes. For example, bacterial (6-4) photolyases, which are at least in some cases capable to act as cryptochromes as well, and class II CPD photolyases (CPDII) differ from eukaryotic (6-4) photolyases and other CPD photolyases in terms of DNA recognition, electron transfer pathways and auxillary photoantennas. We will explore DNA repair catalyzed by bacterial (6-4) photolyases by bundling our competences structural biology, photobiology and time-resolved spectroscopy. Here, we will first focus on PhrB from Agrobacterium tumefaciens and other well-established members of the family of bacterial (6-4) photolyases. To address their mechanisms in terms of flavin photoreduction and 6-4PP repair we employ transient absorption techniques with exceptionally wide ranges of temporal observation (100 fs to seconds) and spectral coverage. Additionally, we will systematically apply site-directed mutagenesis to fine-map electron and proton transfer reactions during 6-4PP repair to reveal otherwise obscured reaction pathways. The mode of 6-4PP-DNA binding to bacterial (6-4) photolyases and the effect of mutations on the 3D structure will be analyzed by X-ray crystallography. Concise mechanistic models consistent with our spectroscopic and structural data will be delineated for this type of (6-4) photolyases and may have profound influence on our understanding of eukaryotic (6-4) photolyases as well. Finally, the evolutionary origin of the PCSf is currently still unresolved. Using our structural and mechanistic knowledge on members of the PCSf we will characterize a novel PCSf family with predicted CPD photolyase activity. Members of this family lack the N-terminal antenna domain otherwise strictly conserved within the PCSf. In parallel, we will pursue a reverse-engineering strategy to generate and characterize minimal single-domain photolyases from known PCSf members. This approach will address the protein evolution of the PCSf and may lead us to the earliest ancestors of the PCSf, which may not have yet required an additional blue-light harvesting antenna domain and depended entirely on the photochemistry of their FADH(−) chromophore.