TY - JOUR

T1 - Pre-steady-state kinetic analysis of dNTP and DNA binding by human terminal deoxynucleotidyl transferase

AU - Senchurova, Svetlana S.

AU - Kuznetsova, Aleksandra A.

AU - Kuznetsov, Nikita A.

N1 - Svetlana S. Senchurova, Aleksandra A. Kuznetsova, Nikita A. Kuznetsov, Pre-steady-state kinetic analysis of dNTP and DNA binding by human terminal deoxynucleotidyl transferase, International Journal of Biological Macromolecules, Volume 383, Part 2, 2026, 154663, ISSN 0141-8130, https://doi.org/10.1016/j.ijbiomac.2026.154663. This work was funded by the Russian Science Foundation grant number 21-64-00017p. Partial support by Russian state-funded project No. 125012300658-9 for the routine maintenance of the equipment is also acknowledged.

PY - 2026/11

Y1 - 2026/11

N2 - 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 (Cpy) 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.

AB - 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 (Cpy) 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.

KW - Functional role of active site amino acids

KW - Kinetics of dNTP and primer binding

KW - Pre-steady-state kinetics

KW - Terminal deoxynucleotidyl transferase

KW - Терминальная дезоксинуклеотидилтрансфераза

KW - Кинетика связывания дНТФ и праймера

KW - Кинетика в предстационарном состоянии

KW - Функциональная роль аминокислот активного центра

UR - https://www.mendeley.com/catalogue/0793a8f6-9609-3a7e-ba61-b26c686cf33a/

UR - https://www.scopus.com/pages/publications/105052141487

U2 - 10.1016/j.ijbiomac.2026.154663

DO - 10.1016/j.ijbiomac.2026.154663

M3 - Article

C2 - 42805534

VL - 383

JO - International Journal of Biological Macromolecules

JF - International Journal of Biological Macromolecules

SN - 1879-0003

M1 - 154663

ER -