Impact of alpha-hydroxy-propanodeoxyguanine adducts on DNA duplex energetics: opposite base modulation and implications for mutagenicity and genotoxicity

Abstract

Acrolein is an alpha,beta-unsaturated aldehyde that is a major environmental pollutant, as well as a product of cellular metabolism. DNA bases react with acrolein to form two regioisomeric exocyclic guanine adducts, namely gamma-hydroxy-propanodeoxyguanosine (gamma-OH-PdG) and its positional isomer alpha-hydroxy-propanodeoxyguanosine (alpha-OH-PdG). The gamma-OH-PdG isomer adopts a ring-opened conformation with minimal structural perturbation of the DNA host duplex. Conversely, the alpha-OH-PdG isomer assumes a ring-closed conformation that significantly disrupts Watson-Crick base-pair alignments within the immediate vicinity of the damaged site. We have employed a combination of calorimetric and spectroscopic techniques to characterize the thermodynamic origins of these lesion-induced structural alterations. Specifically, we have assessed the energetic impact of alpha-OH-PdG centered within an 11-mer duplex by hybridizing the adduct-containing oligonucleotide with its complementary strand harboring a central base N [where N = C or A], yielding a pair of duplexes containing the nascent lesion (alpha-OH-PdG.C) or mismatched adduct (alpha-OH-PdG.A), respectively. Our data reveal that the nascent lesion is highly destabilizing, whereas its mismatched counterpart partially ameliorates alpha-OH-PdG-induced destabilization. Collectively, our data provide energetic characterizations of the driving forces that modulate error-free versus error-prone DNA translesion synthesis. The biological implications of our findings are discussed in terms of energetically probing acrolein-mediated mutagenicity versus adduct-induced genotoxicity.

Figure 1. Chemical Structures of Acrolein Adducts and DNA Host Duplexes

A. The regioisomers of hydroxy-propanodeoxyguanosine designated as α-OH-PdG (I) and γ-OH-PdG (II) compared with the stable analog PdG (III). B. The exocyclic damaged G (α-OH-PdG) represented by G* is embedded as the central base within an 11-mer deoxyoligonucleotide that is hybridized with ^5’GCATGCGTACG^3’ or ^5’GCATGAGTACG^3’ to yield the α-OH-PdG·C or α-OH-PdG·A duplexes, respectively. The 11-mer host duplexes designated as the G·C Parent and G·A Mismatch appear on the left, while the damaged α-OH-PdG·C and α-OH-PdG·A duplexes designated as G*C and G*A appear on the right.

Figure 2. Circular Dichroism Spectra of Duplexes Harboring the Acrolein Adduct

Comparison of normalized circular dichroism spectra expressed in the form of molar ellipticity for the α-OH-PdG·C (red) and α-OH-PdG·A (magenta) duplexes relative to the corresponding G·C Parent (blue) and G·A Mismatch (navy) 11-mers.

Figure 3. Impact of an Acrolein Adduct on Thermodynamic Stability of the DNA Host Duplex

Comparison of excess heat capacity profiles determined for the α-OH-PdG·C (red) and α-OH-PdG·A (magenta) duplexes relative to the corresponding G·C parent (blue) and G·A mismatch (navy) 11-mers. The inset furnishes van’t Hoff analyses of the optically-derived concentration-dependent dissociation profiles for these four duplexes. The “undamaged” G·A mismatch and adduct-containing 11-mers paired with dA or dC significantly disrupt cooperative duplex dissociation with differential destabilization ranked as follows: G·A < α-OH-PdG·A < α-OH-PdG·C.

Figure 4. Impact of α-OH-PdG and Opposite Base on non-ΔC_p-corrected (light blue) and ΔC_p-corrected (dark blue) Thermodynamic Parameters

Differential thermodynamic destabilization of the G·C Parent sorted by decreasing order of energetic impact via replacement of dG by α-OH-PdG and/or dC by dA as specifically noted. The adduct-induced impacts on dissociation free energies are expressed as - ΔΔG to improve clarity and sorted on the basis of decreasing ΔC_p-corrected ΔΔG. Replacement of dC with dA alleviates the overall energetic impact of α-OH-PdG irrespective of ΔC_p-corrections, as noted by the observation that - ΔΔG < 0 for α-OH-PdG·C → α-OH-PdG·A.

Figure 5. Schematic Representation Illustrating the Fate of a Damaged dG within Genomic DNA

Following exposure to a damaging agent, a nascent lesion may be removed by the cellular repair machinery as depicted in the vertical pathway on the left. When an egregious lesion escapes repair, the resultant damaged strand undergoes replication via one of the following horizontal pathways: (A) Error-free synthesis resulting in restoration of the canonical duplex: (B) Blocking synthesis that stalls within the vicinity of the lesion site, resulting in DNA synthesis arrest and consequent genotoxicity; or, (C) Error-prone synthesis via nucleotide misincorporation, forming a mutagenic intermediate that yields a mutant product (e.g., G to T substitution).

Figure 6. Lesion-Induced Energetic Impacts as a Probe of Cytotoxicity and Mutagenicity

Comparison of the G to G* modification within the “nascent” and mismatch duplexes as monitored by the ΔΔG of G·C → G*·C (burgundy) versus G·A → G*·A (blue) for the α-OH-PdG, PdG, and 8-oxodG lesions. Although the thermodynamic parameters are derived from DNA duplexes of distinct sequence context under different solution conditions, each data set is obtained via direct comparison with its respective reference duplex (i.e., ΔΔG_{GC to G*C} = ΔG_G*C − ΔG_GC and ΔΔG_{GA to G*A} = ΔG_G*A − ΔG_GA). The lesion-induced impacts on duplex dissociation free energies are expressed as - ΔΔG to improve clarity and sorted on the basis of decreasing ΔΔG. Amongst these lesion-containing duplexes, 8-oxodG·A is thermodynamically stabilized relative to the corresponding “undamaged” G·A mismatch.