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. 2012 Oct;40(18):9193-205.
doi: 10.1093/nar/gks653. Epub 2012 Jul 5.

Preferred WMSA catalytic mechanism of the nucleotidyl transfer reaction in human DNA polymerase κ elucidates error-free bypass of a bulky DNA lesion

Lee Lior-Hoffmann  1 , Lihua Wang, Shenglong Wang, Nicholas E Geacintov, Suse Broyde, Yingkai Zhang
Affiliations

Preferred WMSA catalytic mechanism of the nucleotidyl transfer reaction in human DNA polymerase κ elucidates error-free bypass of a bulky DNA lesion

Lee Lior-Hoffmann et al. Nucleic Acids Res. 2012 Oct.

Abstract

Human DNA Pol κ is a polymerase enzyme, specialized for near error-free bypass of certain bulky chemical lesions to DNA that are derived from environmental carcinogens present in tobacco smoke, automobile exhaust and cooked food. By employing ab initio QM/MM-MD (Quantum Mechanics/Molecular Mechanics-Molecular Dynamics) simulations with umbrella sampling, we have determined the entire free energy profile of the nucleotidyl transfer reaction catalyzed by Pol κ and provided detailed mechanistic insights. Our results show that a variant of the Water Mediated and Substrate Assisted (WMSA) mechanism that we previously deduced for Dpo4 and T7 DNA polymerases is preferred for Pol κ as well, suggesting its broad applicability. The hydrogen on the 3'-OH primer terminus is transferred through crystal and solvent waters to the γ-phosphate of the dNTP, followed by the associative nucleotidyl transfer reaction; this is facilitated by a proton transfer from the γ-phosphate to the α,β-bridging oxygen as pyrophosphate leaves, to neutralize the evolving negative charge. MD simulations show that the near error-free incorporation of dCTP opposite the major benzo[a]pyrene-derived dG lesion is compatible with the WMSA mechanism, allowing for an essentially undisturbed pentacovalent phosphorane transition state, and explaining the bypass of this lesion with little mutation by Pol κ.

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Figures

Figure 1.
Figure 1.
Structure of the 10S (+)-trans-anti-B[a]P-N2-dG (B[a]P-dG) adduct. The absolute configurations of the four chiral atoms C7, C8, C9 and C10 are indicated. Torsion angles are defined as χ = O4′(dR) – C1′(dR) – N9 – C4 (dR is deoxyribose), α′ = N1 – C2 – N2 – C10(B[a]P-dG), β′ = C2 – N2 – C10(B[a]P-dG) – C9(B[a]P-dG).
Figure 2.
Figure 2.
Active site of Pol κ ternary complex ((32) and PDB ID: 2OH2) remodeled with MD as described in Computational Methods section. This structure was utilized to initiate the QM/MM–MD simulations. The octahedral coordination of the two Mg+2 ions is shown with the dashed lines and their distances are shown in Supplementary Figure S2. Hydrogen atoms are not displayed for clarity except for the H(O3′) and the crystal water hydrogens.
Scheme 1.
Scheme 1.
Computational protocol.
Figure 3.
Figure 3.
WMSA mechanism: (A, B) Reaction schemes and (C) Free energy profile. R: reactant; TS1: transition state 1; I: intermediate; TS2: transition states 2; P: product. These are illustrated in Figure 4.
Figure 4.
Figure 4.
Key structures of the nucleotidyl transfer reaction catalyzed by Pol κ: reaction mechanism (left) and critical structures (right). See Supplementary Movies S1 and S2 for mechanism details.
Figure 5.
Figure 5.
Key stabilizing hydrogen-bonding interactions (dashed lines) between active site amino acid residues and the pentacovalent phosphorane transition state. (A) Unmodified system simulated with QM/MM–MD (B) Modified system simulated with classical MD. The unmodified system simulated with classical MD preserves the same hydrogen bonding interactions as shown in Supplementary Table S3.
Figure 6.
Figure 6.
The α, β and γ phosphate group charges during key stationary points of the reaction (Figure 4). The partial charge of the bridging oxygen atoms is considered to be evenly shared between the neighboring phosphate groups.
Figure 7.
Figure 7.
Stabilizing interactions (electrostatic and van der Waals) between Pol κ amino acid residues and the QM active site region (defined in Supplementary Figure S3) for the attack-ready O3′ intermediate and the pentacovalent phosphorane transition state (Figure 4). See also Supplementary Figures S9 and S10.
Figure 8.
Figure 8.
Free energy profile of the torsion angle C2′–C3′–O3′–H determining the orientation of the H(O3′). At the 45° minimum the hydrogen is oriented towards the α-phosphate while at the 309° minimum it is directed toward E199.
Figure 9.
Figure 9.
Pol κ containing the B[a]P-dG adduct in the minor groove at the pentacovalent phosphorane transition state. View is towards the major groove and emphasizes the maintained Watson–Crick base pairing between B[a]P-dG and the incoming dCTP. (A) Zoom-out view (B) Close-up view. Color scheme: N-clasp domain, blue; B[a]P ring system, red; lesion-containing guanine, cyan; Primer-linked dCTP, pink. Watson–Crick hydrogen bonds between B[a]P-dG and dCTP, black dashed lines. The pentacovalent phosphorane is indicated by an arrow. See Supplementary Movie S3.
Figure 10.
Figure 10.
Pol κ containing the B[a]P-dG adduct viewed into the minor groove in close-up (A) Prior to reaction with the O3′H designated by an orange circle (B) At the transition state with pentacovalent phosphorane designated by an orange circle; Color scheme: N-clasp domain, blue; B[a]P ring system, red; lesion-containing guanine, cyan; dCTP and the primer are colored by atom. See Supplementary Movie S3.
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