Kinetic and Structural Impact of Metal Ions and Genetic Variations on Human DNA Polymerase ι

Abstract

DNA polymerase (pol) ι is a Y-family polymerase involved in translesion synthesis, exhibiting higher catalytic activity with Mn²⁺ than Mg²⁺ The human germline R96G variant impairs both Mn²⁺-dependent and Mg²⁺-dependent activities of pol ι, whereas the Δ1-25 variant selectively enhances its Mg²⁺-dependent activity. We analyzed pre-steady-state kinetic and structural effects of these two metal ions and genetic variations on pol ι using pol ι core (residues 1-445) proteins. The presence of Mn²⁺ (0.15 mm) instead of Mg²⁺ (2 mm) caused a 770-fold increase in efficiency (k_pol/K_d,dCTP) of pol ι for dCTP insertion opposite G, mainly due to a 450-fold decrease in K_d,dCTP The R96G and Δ1-25 variants displayed a 53-fold decrease and a 3-fold increase, respectively, in k_pol/K_d,dCTP for dCTP insertion opposite G with Mg²⁺ when compared with wild type, substantially attenuated by substitution with Mn²⁺ Crystal structures of pol ι ternary complexes, including the primer terminus 3'-OH and a non-hydrolyzable dCTP analogue opposite G with the active-site Mg²⁺ or Mn²⁺, revealed that Mn²⁺ achieves more optimal octahedral coordination geometry than Mg²⁺, with lower values in average coordination distance geometry in the catalytic metal A-site. Crystal structures of R96G revealed the loss of three H-bonds of residues Gly-96 and Tyr-93 with an incoming dNTP, due to the lack of an arginine, as well as a destabilized Tyr-93 side chain secondary to the loss of a cation-π interaction between both side chains. These results provide a mechanistic basis for alteration in pol ι catalytic function with coordinating metals and genetic variation.

Keywords: DNA enzyme; DNA polymerase; X-ray crystallography; crystal structure; enzyme kinetics; genetic variation; kinetics; metal ion-protein interaction; pre-steady-state kinetics; translesion DNA synthesis.

FIGURE 1.

Structures of pol ι·DNA·nucleotide ternary complexes in the presence of Mg²⁺ or Mn²⁺. A, superposition of the overall structures of ternary complexes of pol ι(26–445) with DNA containing template G and incoming non-hydrolyzable dCMPNPP in the presence of Mg²⁺ (blue) or Mn²⁺ (red). pol ι is shown as ribbons, DNA is shown as tube and ladder, the nucleotide is shown as sticks, and metal ions are shown as spheres. B, superposition of the active sites of the pol ι(26–445)·G·dCMPNPP·Mg²⁺ (blue) and pol ι(26–445)·G·dCMPNPP·Mn²⁺ (red) complexes. Active-site residues, DNA, and nucleotide are shown as sticks, and metal ions are shown as spheres.

FIGURE 2.

Comparison of Mg²⁺ and Mn²⁺ coordinations in the pol ι active site. A and B, F_o − F_c simulated annealing omit maps (blue) contoured at 3.0σ for the incoming dCMPNPP and Mg²⁺ (A) or Mn²⁺ (B) ions in the active sites of pol ι. The electron density is superimposed on the refined modeled dCMPNPP and metal ions. Mg²⁺ and Mn²⁺ ions are shown as green and magenta spheres, respectively. C and D, close-up view of Mg²⁺ (C) or Mn²⁺ (D) coordination in the active-site metal binding sites of pol ι·G·dCMPNPP complexes. Metal ion coordination is shown as dashed lines, and the coordination distances are indicated. E, close-up view of Mg²⁺ coordination in the active-site metal binding site of pol η·G·dCMPNPP complex (PDB ID 4DL3). Mg²⁺ ions are shown as gray spheres. F, superposition of the active-site metal binding sites of the pol ι(26–445)·G·dCMPNPP·Mg²⁺ (green), pol ι(26–445)·G·dCMPNPP·Mn²⁺ (magenta), and pol η·G·dCMPNPP·Mg²⁺ (PDB ID 4DL3, gray) complexes. Red, blue, and orange colors indicate oxygen, nitrogen, and phosphorus atoms, respectively. Cyan (in A and C), light blue (in B and D), and light gray (in E) colors indicate carbon atoms.

FIGURE 3.

The active sites of R96G pol ι ternary complexes in the presence of Mg²⁺ or Mn²⁺. A–D, F_o − F_c simulated annealing omit maps contoured at 3.0σ for the residues Arg-96 (or Gly-96) and Tyr-93 in the active sites of pol ι(26–445)·G·dCMPNPP·Mg²⁺ (A); R96G pol ι(1–445)·G·dCMPNPP·Mg²⁺ (B); pol ι(26–445)·G·dCMPNPP·Mn²⁺ (C); and R96G pol ι(1–445)·G·dCMPNPP·Mn²⁺ (D) complexes. The electron density (blue mesh) is superimposed on the refined modeled residues Arg-96 (or Gly-96) and Tyr-93. Mg²⁺ and Mn²⁺ ions are shown as green and magenta spheres. E–H, close-up view of the active sites of pol ι(26–445)·G·dCMPNPP·Mg²⁺ (D); R96G pol ι(1–445)·G·dCMPNPP·Mg²⁺ (F); pol ι(26–445)·G·dCMPNPP·Mn²⁺ (G); and R96G pol ι(1–445)·G·dCMPNPP·Mn²⁺ (H) complexes. Hydrogen-bonding interactions are shown as dashed lines. Red, blue, orange, and yellow colors indicate oxygen, nitrogen, phosphorus, and sulfur atoms, respectively. Cyan (in A, C, E, and G) and light pink (in B, D, F, and H) colors indicate carbon atoms.