TY - JOUR

T1 - Insight into the mechanism of DNA synthesis by human terminal deoxynucleotidyltransferase

AU - Kuznetsova, Aleksandra A.

AU - Tyugashev, Timofey E.

AU - Alekseeva, Irina V.

AU - Timofeyeva, Nadezhda A.

AU - Fedorova, Olga S.

AU - Kuznetsov, Nikita A.

N1 - Publisher Copyright: © 2022 Kuznetsova et al.

PY - 2022/12/1

Y1 - 2022/12/1

N2 - Terminal deoxynucleotidyltransferase (TdT) is a member of the DNA polymerase X family that is responsible for random addition of nucleotides to single-stranded DNA. We present investigation into the role of metal ions and specific interactions of dNTP with active-site amino acid residues in the mechanisms underlying the recognition of nucleoside triphosphates by human TdT under pre-steady-state conditions. In the elongation mode, the ratios of translocation and dissociation rate constants, as well as the catalytic rate constant were dependent on the nature of the nucleobase. Preferences of TdT in dNTP incorporation were researched by molecular dynamics simulations of complexes of TdT with a primer and dNTP or with the elongated primer. Purine nucleotides lost the "summarised" H-bonding network after the attachment of the nucleotide to the primer, whereas pyrimidine nucleotides increased the number and relative lifetime of H-bonds in the post-catalytic complex. The effect of divalent metal ions on the primer elongation revealed that Me2+ cofactor can significantly change parameters of the primer elongation by strongly affecting the rate of nucleotide attachment and the polymerisation mode.

AB - Terminal deoxynucleotidyltransferase (TdT) is a member of the DNA polymerase X family that is responsible for random addition of nucleotides to single-stranded DNA. We present investigation into the role of metal ions and specific interactions of dNTP with active-site amino acid residues in the mechanisms underlying the recognition of nucleoside triphosphates by human TdT under pre-steady-state conditions. In the elongation mode, the ratios of translocation and dissociation rate constants, as well as the catalytic rate constant were dependent on the nature of the nucleobase. Preferences of TdT in dNTP incorporation were researched by molecular dynamics simulations of complexes of TdT with a primer and dNTP or with the elongated primer. Purine nucleotides lost the "summarised" H-bonding network after the attachment of the nucleotide to the primer, whereas pyrimidine nucleotides increased the number and relative lifetime of H-bonds in the post-catalytic complex. The effect of divalent metal ions on the primer elongation revealed that Me2+ cofactor can significantly change parameters of the primer elongation by strongly affecting the rate of nucleotide attachment and the polymerisation mode.

KW - DNA Nucleotidylexotransferase/chemistry

KW - DNA Replication

KW - DNA, Single-Stranded

KW - Humans

KW - Ions

KW - Nucleotides

UR - http://www.scopus.com/inward/record.url?scp=85135306187&partnerID=8YFLogxK

U2 - 10.26508/lsa.202201428

DO - 10.26508/lsa.202201428

M3 - Article

C2 - 35914812

AN - SCOPUS:85135306187

VL - 5

JO - Life science alliance

JF - Life science alliance

SN - 2575-1077

IS - 12

M1 - e202201428

ER -