Insight into the mechanism of DNA synthesis by human terminal deoxynucleotidyltransferase

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.

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.

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.

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.

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.

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.

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.

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.