TY - JOUR

T1 - Substrate Specificity Diversity of Human Terminal Deoxynucleotidyltransferase May Be a Naturally Programmed Feature Facilitating Its Biological Function

AU - Kuznetsova, Aleksandra A

AU - Senchurova, Svetlana I

AU - Gavrilova, Anastasia A

AU - Tyugashev, Timofey E

AU - Mikushina, Elena S

AU - Kuznetsov, Nikita A

N1 - This work was supported partially by a Russian-Government-funded project (No. 121031300041-4). The part of this work involving experimental data analysis was specifically funded by Russian Science Foundation grant number 21-64-00017.

PY - 2024/1

Y1 - 2024/1

N2 - 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 Mg2+ ions. Surrogate metal ions Co2+ or Mn2+ 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.

AB - 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 Mg2+ ions. Surrogate metal ions Co2+ or Mn2+ 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.

KW - Humans

KW - DNA Nucleotidylexotransferase

KW - Substrate Specificity

KW - DNA-Directed DNA Polymerase

KW - Catalysis

KW - Coloring Agents

KW - Nucleotides

KW - Ions

UR - https://www.scopus.com/record/display.uri?eid=2-s2.0-85183220074&origin=inward&txGid=9adedf2a77f6d0a3a2e5ac531d599ec2

U2 - 10.3390/ijms25020879

DO - 10.3390/ijms25020879

M3 - Article

C2 - 38255952

VL - 25

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1661-6596

IS - 2

M1 - 879

ER -