Characterization of deoxyribonuclease (DNase) activity in DNA-fed starved Tetrahymena thermophila cells based on gene expression

Deoxyribonucleases (DNases) are a group of enzyme that breaks down the single or double stranded DNA molecules in a non-specific sequence manner. These enzymes have myriad biological functions in vivo for instance DNA fragmentation in apoptosis and being defense barrier for the exogenous DNA uptake. In vitro, during routine laboratory works, it is necessary to use a DNase to prevent DNA contamination in protein and RNA isolations. Tetrahymena thermophila, a unicellular eukaryotic ciliate, is a well-known model organism which is widely used for regulatory, system and molecular biology studies; however, there is no extensive DNase study in this organism. Its life cycle consists of three stages namely, vegetative, starvation and conjugation. Due to the increasing potential of being a eukaryotic protein expression host in recent years, it is found that characterization of DNase activity in Tetrahymena is crucial. So far as, DNase activity is a parameter that makes the transformation of an organism more difficult. In this study, bioinformatics analyses showed that Tetrahymena genome encodes five DNase II genes. All of these proteins possess catalytic TxDHSKW motif, which is specific for DNase II proteins. Phylogenetic analyses also showed that Tetrahymena DNase II proteins have ancestral characteristics comparing to higher eukaryotic DNases. Experimentally, characterization of DNase activity of vegetative and starved cells was performed with agarose and zymogram assays by using whole cell extracts. As a result of zymography, a total of five DNase activity bands, ranging between 28-69 kDa, were identified. Highest DNase activity was obtained at acidic pH (pH 5) and Mg2+ ions inhibit neutral DNase activities significantly. DNA treatment of starved cells partially increased extracellular DNase activity of a 32 kDa protein. As a result, the findings of this study will help to simplify the usage of Tetrahymena as a eukaryotic recombinant protein expression host. Key words: Tetrahymena, DNase activity, DNase II, DNA-treatment.