Structural and functional analysis of Sulfolobus solfataricus Y-family DNA polymerase Dpo4-catalyzed bypass of the malondialdehyde-deoxyguanosine adduct

Abstract

Oxidative stress can induce the formation of reactive electrophiles, such as DNA peroxidation products, e.g., base propenals, and lipid peroxidation products, e.g., malondialdehyde. Base propenals and malondialdehyde react with DNA to form adducts, including 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10(3H)-one (M1dG). When paired opposite cytosine in duplex DNA at physiological pH, M1dG undergoes ring opening to form N2-(3-oxo-1-propenyl)-dG (N2-OPdG). Previous work has shown that M1dG is mutagenic in bacteria and mammalian cells and that its mutagenicity in Escherichia coli is dependent on induction of the SOS response, indicating a role for translesion DNA polymerases in the bypass of M1dG. To probe the mechanism by which translesion polymerases bypass M1dG, kinetic and structural studies were conducted with a model Y-family DNA polymerase, Dpo4 from Sulfolobus solfataricus. The level of steady-state incorporation of dNTPs opposite M1dG was reduced 260-2900-fold and exhibited a preference for dATP incorporation. Liquid chromatography-tandem mass spectrometry analysis of the full-length extension products revealed a spectrum of products arising principally by incorporation of dC or dA opposite M1dG followed by partial or full-length extension. A greater proportion of -1 deletions were observed when dT was positioned 5' of M1dG. Two crystal structures were determined, including a "type II" frameshift deletion complex and another complex with Dpo4 bound to a dC.M1dG pair located in the postinsertion context. Importantly, M1dG was in the ring-closed state in both structures, and in the structure with dC opposite M1dG, the dC residue moved out of the Dpo4 active site, into the minor groove. The results are consistent with the reported mutagenicity of M1dG and illustrate how the lesion may affect replication events.

Figure 1

Schematic illustration of pathways generating M₁dG/N²-OPdG and the equilibrium between the ring-open and ring-closed forms of the lesion.

Figure 2

Pre-steady-state analysis of Dpo4-catalyzed incorporation opposite M₁dG. (A) Dpo4 (350 nM) was incubated with radiolabeled 18/23-mer DNA (200 nM), the indicated dNTP (1 mM), and MgCl₂ (5 mM). The data were fit to eq 1 to yield the following kinetic parameters: for dCTP·G (◼), A = 170 ± 9 nM and k_obs = 8.9 ± 2.3 s⁻¹; for dCTP·M₁dG (blue ●), A = 15 ± 2 nM and k_obs = 0.81 ± 0.35 s⁻¹; for dATP·M₁dG (red ▲), A = 95 ± 4 nM and k_obs = 0.051 ± 0.004 s⁻¹. (B) The results from panel A are shown to illustrate the small amount of product formed rapidly during dCTP insertion opposite M₁dG.

Figure 3

LC−MS analysis of Dpo4-catalyzed full-length extension products. (A) ESI mass spectrum of products derived from Dpo4-catalyzed extension of 13/18-mer DNA containing M₁dG with cytosine to the 5′ side of the lesion. The products identified in the spectrum are summarized. (B) ESI mass spectrum of products derived from Dpo4-catalyzed extension of 13/18-mer DNA containing M₁dG with thymidine to the 5′ side of the lesion. The products identified in the spectrum are summarized.

Figure 4

Configuration of bases in the active site of Dpo4·M₁dG ternary complexes. (A) The quality of the electron density from the dGTP·M₁dG crystal structure is shown (gray wire mesh) with the 3F_o − 2F_c map contoured to 1σ. Dpo4 (cyan) is shown in schematic form in complex with primer−/template DNA (yellow carbons) and calcium ions (blue spheres). (B) The quality of the electron density from the 14C·M₁dG crystal structure is shown (gray wire mesh) with the 3F_o − 2F_c map contoured to 1σ. Dpo4 (green) is shown in schematic form in complex with primer−template DNA (yellow carbons) and calcium ions (blue spheres). (C) The orientation of the primer terminus (14C) relative to M₁dG from the 14C·M₁dG stucture is shown as viewed from the palm domain of Dpo4. (D) Stacking interactions between the DNA residues in both structures are shown. The C·G pair at the primer terminus (blue) is shown in the background, with M₁dG and the dGTP·C pair (green) stacked in the foreground along the helical axis. The 14th primer residue from the 14C·M₁dG crystal structure (14C) is colored yellow. The M₁dG moiety is colored red in all panels for the sake of clarity.