Tek hücreli organizmalarda bulunan kriptokrom proteininin yapı-işlev ilişkisinin araştırılması

Photoreceptors allow living organisms to gain information about their external world and thus adapt themselves and optimize their metabolism to changes in the nature. The photolyase/cryptochrome (Phr/Cry) family is a large and diversified gene family that encodes DNA repair enzymes and blue-light photoreceptors. They exist widespread in three kingdoms of life. There are 3 major categories of proteins represented by this family: 1) The cyclobutane pyrimidine dimer (CPD) Phr, 2) the [6-4] Phr, and 3) the cryptochromes (Cry). Photolyases are enzymes that utilize light energy for repair of UV lesion on DNA. Cryptochromes, in contrast, function as signaling molecules that regulate diverse biological responses such as entrainment of circadian rhythms in plants and animals. Nevertheless, the role of the Crys is ill-defined especially in unicellular organisms. From the evolutionary point of view, the Crys in such lower organisms hold the potential to be the ancestors of animal and plant cryptochromes. Hence, identification of new members of Phr/Cry family encoded by the genomes of different unicellular organisms is important to understand functional diversity of Cry family and also to know how structure influences cryptochrome function. Towards this aim, this dissertation concentrates on the analysis of structure-function and kinetics of cryptochromes in unicellular organisms via biochemical complementation and SPR spectroscopy methods. Here, seven new members of the PHR/CRY family of a red algae Cyanidioschyzon merolae were identified. Evolutionary relationships of algal Phr-like genes revealed that one gene is close to the [6-4] photolyase, three to the single strand DNA repairing subfamily of cryptochromes; called Cry-DASHes. Also, two plant type cryptochromes and one blue light photoreceptor as an independent clade between photolyase and plant type cryptochrome were found. Both, biochemical and complementation data provide evidence that C. merolae harbors two functional Cry-DASHes in addition to the one without DNA repair property. Functional characterization of photolyase/cryptochrome family from such a primitive algae provides insights into the evolution and the origin of plant cryptochrome lineages back to early eukaryotes. Secondly, the repair kinetics of a VcCry-DASH and a VcPhr were investigated using Surface Plasmon Resonance (SPR). To our knowledge for the first time, we quantified the affinity of VcCry-DASH to ssDNA and dsDNA containing CPD UV lesion. Moreover, we showed that DNA repair assays can be made by SPR spectroscopy by real time monitoring DNA repair.?