Abstract
Terminal 2'-deoxynucleotidyl transferase (TdT) is a unique enzyme capable of catalysing template-independent elongation of DNA 3' ends during V(D)J recombination. The mechanism controlling the enzyme's substrate specificity, which is necessary for its biological function, remains unknown. Accordingly, in this work, kinetic and mutational analyses of human TdT were performed and allowed to determine quantitative characteristics of individual stages of the enzyme-substrate interaction, which overall may ensure the enzyme's operation either in the distributive or processive mode of primer extension. It was found that conformational dynamics of TdT play an important role in the formation of the catalytic complex. Meanwhile, the nature of the nitrogenous base significantly affected both the dNTP-binding and catalytic-reaction efficiency. The results indicated that neutralisation of the charge and an increase in the internal volume of the active site caused a substantial increase in the activity of the enzyme and induced a transition to the processive mode in the presence of Mg(2+) ions. Surrogate metal ions Co(2+) or Mn(2+) also may regulate the switching of the enzymatic process to the processive mode. Thus, the totality of individual factors affecting the activity of TdT ensures effective execution of its biological function.