TY - JOUR

T1 - Coordination of Nucleoside Triphosphates in the Active Site of the Human TdT during Template-Independent DNA Synthesis

AU - Senchurova, Svetlana I.

AU - Tyugashev, Timofey E.

AU - Kuznetsov, Nikita A.

N1 - Senchurova, S. I. Coordination of Nucleoside Triphosphates in the Active Site of the Human TdT during Template-Independent DNA Synthesis / S. I. Senchurova, T. E. Tyugashev, N. A. Kuznetsov // Biochemistry (Moscow). – 2026. – Vol. 91, No. 1. – P. 102-118. – DOI 10.1134/S0006297925602345. – EDN OZKMCI. This work was financially supported by the Russian Science Foundation (grant 21-64-00017-П) and partially by the State Budget project no. 125012300658-9, involving conducting supplementary work and service of equipment used in the study

PY - 2026/2/10

Y1 - 2026/2/10

N2 - Human terminal deoxynucleotidyl transferase (TdT) belongs to the family X DNA polymerases responsible for increasing diversity of immunoglobulins and T-cell receptor genes during V(D)J recombination by means of template-independent addition of random nucleotides to the 3'-end of the rearranged V-D and J-D segments. According to the X-ray structural data, coordination of the incoming dNTP and 3'-terminal nucleotides of the single-stranded primer in the active site of TdT is accomplished through contacts with the hydrophobic (W449, F404, and L397) and hydrophilic (R336, H342, D395, E456, R453, and R457) residues that form the dNTP-binding pocket. The residues R336, H342, and D345 are directly involved in orientation of the phosphate groups of the incoming dNTP and stabilization of the 3'-terminal nucleotide of the DNA primer. In this work, we analyzed the consequences of replacing these residues using molecular dynamics methods and experimentally tested enzymatic properties of the mutant forms of the human TdT containing the R336Q, H342A, and D345E substitutions. The obtained data showed that the H342A substitution increases dissociation constant of the enzyme complex with dNTP and DNA primer and significantly reduces efficiency of the dNTP addition to the growing chain. Extension of the side chain at the D345 residue disrupts coordination of the cofactor metal ions and also affects efficiency of the catalytic reaction. Substitution of the R336 residue leads to significant destabilization of the protein globule and complete loss of the catalytic activity of the enzyme.

AB - Human terminal deoxynucleotidyl transferase (TdT) belongs to the family X DNA polymerases responsible for increasing diversity of immunoglobulins and T-cell receptor genes during V(D)J recombination by means of template-independent addition of random nucleotides to the 3'-end of the rearranged V-D and J-D segments. According to the X-ray structural data, coordination of the incoming dNTP and 3'-terminal nucleotides of the single-stranded primer in the active site of TdT is accomplished through contacts with the hydrophobic (W449, F404, and L397) and hydrophilic (R336, H342, D395, E456, R453, and R457) residues that form the dNTP-binding pocket. The residues R336, H342, and D345 are directly involved in orientation of the phosphate groups of the incoming dNTP and stabilization of the 3'-terminal nucleotide of the DNA primer. In this work, we analyzed the consequences of replacing these residues using molecular dynamics methods and experimentally tested enzymatic properties of the mutant forms of the human TdT containing the R336Q, H342A, and D345E substitutions. The obtained data showed that the H342A substitution increases dissociation constant of the enzyme complex with dNTP and DNA primer and significantly reduces efficiency of the dNTP addition to the growing chain. Extension of the side chain at the D345 residue disrupts coordination of the cofactor metal ions and also affects efficiency of the catalytic reaction. Substitution of the R336 residue leads to significant destabilization of the protein globule and complete loss of the catalytic activity of the enzyme.

KW - Humans

KW - DNA Nucleotidylexotransferase/metabolism

KW - Catalytic Domain

KW - DNA/biosynthesis

KW - Molecular Dynamics Simulation

KW - DNA Replication

KW - терминальная дезоксинуклеотидилтрансфераза человека

KW - белково-нуклеиновые взаимодействия

KW - координация дНТФ

KW - связывание с ДНК

KW - ферментативный катализ

KW - матрично-независимый синтез ДНК

UR - https://www.scopus.com/pages/publications/105029697397

UR - https://www.elibrary.ru/item.asp?id=89059960

U2 - 10.1134/S0006297925602345

DO - 10.1134/S0006297925602345

M3 - Article

C2 - 41702732

VL - 91

SP - 102

EP - 118

JO - Biochemistry (Moscow)

JF - Biochemistry (Moscow)

SN - 0006-2979

IS - 1

ER -