Pre-steady-state kinetic analysis of dNTP and DNA binding by human terminal deoxynucleotidyl transferase

Terminal deoxynucleotidyl transferase (TdT) is a family X DNA polymerase that performs template-independent attachment of nucleotides to the 3'-end of single stranded DNA during V(D)J recombination. Although X-ray structures and site-directed mutagenesis studies have identified the residues that form the active site of TdT and established their general structural and catalytic roles, a quantitative stage-resolved kinetic description of how these residues govern the individual steps of the catalytic cycle such as DNA and dNTP binding, formation of the precatalytic complex, and the chemical step of nucleotide transfer has not previously been obtained. In this study, using two fluorescent nucleoside triphosphates containing 1,N⁶-ethenoadenine (εA) or pyrrollocytosine (C^py) and a DNA primer containing Cpy-nucleotide at the 3'-end, we investigated the conformational dynamics enzyme-substrate complexes using wild type enzyme and set of mutant forms of human TdT. Stepwise kinetic analysis revealed contribution of individual amino acid residues L397, F400, and F404 formed hydrophobic interactions into the active site as well as R336, H342, and D345 residues, which are directly involved in the coordination of the phosphate groups of the incoming dNTP and stabilization of the 3'-terminal nucleotide of the DNA primer. Fast kinetic approach allowed us to quantitatively characterize the contribution of selected residues in the formation of the specific interactions and the correct positioning of dNTP and DNA primer. Taking together, obtained data provide link between function of active site amino acids and kinetics of molecular events within individual stages of the catalytic cycle.