Identification of the cellular role of the Tetrahymena thermophila TtLigII-1 enzyme that the first member of the newly defined ATP-dependent DNA Ligase II ((LİGII) subfamily in invertebrates

DNA ligase enzymes are involved in the protection, repair and genetic diversity of genomes. While ATP-dependent DNA Ligase I, III and IV are found in vertebrates, Ligase III, which is involved in mitochondrial genome replication and repair, is not found in invertebrate eukaryotes. Single-celled ciliated Tetrahymena thermophila, which is under the invertebrate eukaryotes, has a genome of mitochondria, micronucleus (MIC) and macronucleus (MAK) that direct hereditary and somatic events. In sexual reproduction, MAK is formed from MIC by intense genome editing. T. thermophila genome contains two Lig I, one Lig IV and one hypothetical Ligase gene. Within the scope of this thesis; it is aimed to investigate with in silico, in vivo and in vitro methods whether the hypothetical Ligase gene has ligase activity, and whether it can take part in nuclear and mitochondrial genome repair as well as replication. Maximum likelihood phylogenetic tree analysis revealed that this hypothetical ligase enzyme (TtLigII) is actually a member of a new ligase subfamily called Ligase II, which is only found in invertebrate eukaryotic organisms. TtLigII-6xHis enzyme, which was produced recombinantly in T. thermophila and purified affinitically, has the repair activity of phosphodiester nicks in dsDNA fragments showed by ligating these fragments in in vitro DNA ligase activity reaction. TtLigII-sfGFP was localized into mitochondria, MIC and developing MAC in conjugation. This thesis study concluded that the eukaryotic ATP-dependent DNA Ligase family has a new subfamily called LigII next to the well studied LigI, LigIII and LigIV subfamilies and that its first member, TtLigII, could possibly take role in the repair of nuclei and mitochondria dsDNA nicks.