Insight into the mechanism of DNA synthesis by human terminal deoxynucleotidyltransferase

Abstract

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 Me<sup>2+</sup> cofactor can significantly change parameters of the primer elongation by strongly affecting the rate of nucleotide attachment and the polymerisation mode.

Figure 1.. The two-metal ion mechanism of a polymerase reaction catalysed by well-studied template-dependent DNA polymerases.

Figure 2.. Binding sites of metals A, B, and C in a murine TdT complex with single-stranded DNA and dAMPcPP in the presence of Zn²⁺ (PDB ID: 4I2H).

Figure 3.. The common kinetic mechanism of nucleotide binding and incorporation by DNA polymerase.

E is the enzyme, dNTP is the incoming nucleotide, and DNA_n is the substrate duplex DNA of length n. In most models, E is the open form of the enzyme, and E′ is the closed form, especially for Pol β. The translocation and dissociation stages are simplified for brevity.

Figure 4.

Influence of monovalent cations (K⁺) and pH on TdT activity. (A, B) PAGE analysis of the elongation products in the presence of dNTP at different KCl concentrations (A) and pH levels (B). Reaction conditions: [TdT] = [primer] = 1.0 μM, [dNTP] = 2.0 μM; the concentration of Mg²⁺ was 5.0 mM; reaction time was 1 min in all cases, except for dCTP, for which results on 5 min reaction time are presented. Concentration of KCl in the reaction mixture was varied in the range 0–300 mM, at pH 8.0; when pH was varied from 6.5 to 8.5, KCl concentration was zero. Ext. products indicate integration of all elongation products.

Figure 5.. PAGE analysis of the elongation products under pre–steady-state conditions.

Reaction conditions: 50 mM Tris–HCl (pH 8.0), [TdT] = [primer] = 1.0 μM, [dNTP] = 2.0 μM, [Me²⁺] = 0.5 ÷ 10 mM; reaction time was 1 min in all cases, except for dCTP in the presence of Mg²⁺, for which results on 5 min reaction time are presented.

Figure 6.

Effects of Mg²⁺ and Mn²⁺ ions on TdT activity. (A) PAGE analysis of the elongation products in the presence of a cofactor (Mg²⁺ or Mn²⁺) as a function of time (A). (B, C) Time courses for the incorporation of dGTP and dATP (B) or dTTP and dCTP (C). Reaction conditions: 50 mM Tris–HCl (pH 8.0), [TdT] = [primer] = 1.0 μM, [dNTP] = 2.0 μM, [Mg²⁺] = 5.0 mM or [Mn²⁺] = 1.0 mM.

Figure 7.. PAGE analysis of the elongation primer products under steady-state conditions.

Reaction conditions: 50 mM Tris–HCl (pH 8.0), 5 mM MgCl₂ or 1 mM MnCl₂ or 1 mM CoCl₂; [TdT] = [primer] = 1.0 μM, [dNTP] = 100.0 μM, reaction time was 1 min.

Figure 8.. MST titration curves characterising the interaction of TdT with a DNA primer or Flu-dUTP in the presence of Mg²⁺ (■, ►), Mn²⁺ (●, ♦), or Co²⁺ (▲).

Figure 9.. PAGE analysis of the primer elongation products in the presence of dNTP at different Mg²⁺/Me²⁺ ions ratios.

Reaction conditions: 50 mM Tris–HCl (pH 8.0), [Mg²⁺] = 5.0 mM, and the concentration of Me²⁺ was varied from 0.1 to 1.0 mM. [TdT] = [primer] = 1.0 μM, [dNTP] = 2.0 μM. Reaction time was 1 min in all cases, except for dCTP, for which results on 5 min reaction time are presented.

Figure 10.

Close-up view of the important contacts for the recognition dNTP in the active site of the TdT complex with DNA primer. (A, B, C, D) Molecular dynamics (MD) structure of the human TdT complex with the primer and dGTP (A), dATP (B), dTTP (C), or dCTP (D). The yellow dashed lines indicate direct contacts between amino acid residues of the active site and incoming dNTPs.

Figure 11.

Close-up view of the important contacts between TdT active site and elongated DNA primer. (A, B, C, D) Molecular dynamics (MD) structure of the human TdT complex with an elongated primer after the addition of guanosine (A), adenosine (B), thymidine (C), or cytosine (D). The yellow dashed lines indicate direct contacts between amino acid residues of the active site and the 3′-end nucleotide of the elongated primer. (E) The bending of the elongated primer’s 3′-end in the case of a guanine-ending elongated primer. The green colour indicates the initial state, the yellow colour denotes an intermediate state, and the magenta colour the final state in the MD trajectory.

Figure 12.. The single-stage binding model used for calculation of stability constant of the complex between the primer or Flu-dUTP and the enzyme.

E is the enzyme, S is primer or dNTP, and E•S is the complex.