Mutagenic nucleotide incorporation and hindered translocation by a food carcinogen C8-dG adduct in Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4): modeling and dynamics studies

Abstract

Bulky carcinogen-DNA adducts commonly cause replicative polymerases to stall, leading to a switch to bypass polymerases. We have investigated nucleotide incorporation opposite the major adduct of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the DinB family polymerase, Dpo4, using molecular modeling and molecular dynamics (MD) simulations. PhIP, the most prevalent heterocyclic aromatic amine formed by cooking of proteinaceous food, is mutagenic in mammalian cells and is implicated in mammary and colon tumors. Our results show that the dG-C8-PhIP adduct can be accommodated in the spacious major groove Dpo4 open pocket, with Dpo4 capable of incorporating dCTP, dTTP or dATP opposite the adduct reasonably well. However, the PhIP ring system on the minor groove side would seriously disturb the active site, regardless of the presence and identity of dNTP. Furthermore, the simulations indicate that dATP and dTTP are better incorporated in the damaged system than in their respective mismatched but unmodified controls, suggesting that the PhIP adduct enhances incorporation of these mismatches. Finally, bulky C8-dG adducts, situated in the major groove, are likely to impede translocation in this polymerase (Rechkoblit et al. (2006), PLoS Biol., 4, e11). However, N2-dG adducts, which can reside on the minor groove side, appear to cause less hindrance when in this position.

Figure 1

(a) Structure and numbering scheme of the dG-C8-PhIP adduct. Torsion angle definitions are as follows: χ, O4′-C1′-N9-C4; α′, N9-C8-[PhIP]N²-[PhIP]C2; β′, C8-[PhIP]N²-[PhIP]C2-[PhIP]N1; γ′, [PhIP]C5-[PhIP]C6-[PhIP]C1′-[PhIP]C2′. (b) dG-C8-PhIP-modified DNA sequence used in G·dNTP. Models are derived from a Type I crystal structure of Dpo4 (PDBID: 1S0M) (16). (c) dG-C8-PhIP-modified DNA sequence used in −1 deletion studies. Models are derived from a Type II crystal structure of Dpo4 (PDBID: 1JXL) (15).

Figure 2

The active sites after 3 ns MD in the G·dNTP models. The Dpo4 DNA polymerase, shown in ribbon representation, is colored as follows: thumb is in dark orange, palm in lime, finger in yellow, and little finger in blue; the DNA ternary complex is shown in stick representation. The primer and template DNA strands are colored in gray, the dCTP is colored in cyan, dATP in magenta, dTTP in marine, dGTP in pink, the template guanine is colored in green and the PhIP moiety is in red. Mg²⁺ ions A and B, shown in spheres representation, are colored in orange.

Figure 3

The active sites after 3 ns MD in −1 deletion models. The protein and two Mg²⁺ ions are colored the same as in Figure 2. The template guanine is colored in purple; the dCTP is in cyan; the template guanine without partner is in green, and the PhIP moiety is in red.

Figure 4

Minor groove positioned adducts in Dpo4. The view is into the minor groove side with the major groove in back. (a) syn-dG-C8-PhIP·anti-dATP after 3 ns MD simulation and (b) anti-dG-N²-BP·syn-dATP as in Figure 3b of (54). The carcinogen-modified guanine is green; the PhIP moiety is red; the BP moiety is brown; the incoming dATP is magenta, and the primer-terminus base pair is yellow. The protein is colored the same as in Figure 2.

Figure 5

Major groove positioned anti-dG-C8-PhIP adducts and minor groove positioned anti-dG-N²-BP adducts in Dpo4. For (a) and (b), the view is into the major groove with the minor groove in back, while for (c) and (d), the view is into the minor groove with the major groove in back. (a) anti-dG-C8-PhIP at the insertion position. (b) Modeled anti-dG-C8-PhIP at the post-insertion position with linkage site torsion angles α′, β′ and γ′ same as in (a); collisions between the PhIP rings and the little finger domain are indicated. (c) anti-dG-N²-BP adduct at insertion position opposite anti-dCTP as in Figure 3a of (54). (d) Modeled anti-dG-N²-BP at the post-insertion position in same linkage conformation as (c). The color code is the same as in Figure 4.