Structural context effects in the oxidation of 8-oxo-7,8-dihydro-2'-deoxyguanosine to hydantoin products: electrostatics, base stacking, and base pairing

Abstract

8-Oxo-7,8-dihydroguanine (OG) is the most common base damage found in cells, where it resides in many structural contexts, including the nucleotide pool, single-stranded DNA at transcription forks and replication bubbles, and duplex DNA base-paired with either adenine (A) or cytosine (C). OG is prone to further oxidation to the highly mutagenic hydantoin products spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) in a sharply pH-dependent fashion within nucleosides. In the present work, studies were conducted to determine how the structural context affects OG oxidation to the hydantoins. These studies revealed a trend in which the Sp yield was greatest in unencumbered contexts, such as nucleosides, while the Gh yield increased in oligodeoxynucleotide (ODN) contexts or at reduced pH. Oxidation of oligomers containing hydrogen-bond modulators (2,6-diaminopurine, N(4)-ethylcytidine) or alteration of the reaction conditions (pH, temperature, and salt) identify base stacking, electrostatics, and base pairing as the drivers of the key intermediate 5-hydroxy-8-oxo-7,8-dihydroguanine (5-HO-OG) partitioning along the two hydantoin pathways, allowing us to propose a mechanism for the observed base-pairing effects. Moreover, these structural effects cause an increase in the effective pK(a) of 5-HO-OG, following an increasing trend from 5.7 in nucleosides to 7.7 in a duplex bearing an OG·C base pair, which supports the context-dependent product yields. The high yield of Gh in ODNs underscores the importance of further study on this lesion. The structural context of OG also determined its relative reactivity toward oxidation, for which the OG·A base pair is ~2.5-fold more reactive than an OG·C base pair, and with the weak one-electron oxidant ferricyanide, the OG nucleoside reactivity is >6000-fold greater than that of OG·C in a duplex, leading to the conclusion that OG in the nucleoside pool should act as a protective agent for OG in the genome.

Figure 1

Base pairing schemes for the OG(anti)•C(anti) and OG(syn)•A(anti) base pairs.

Figure 2

18-mer ODN sequences utilized to study context effects on OG oxidation to hydantoin products. In these sequences O = OG and N = A, C, D, F, or ^4EtC. The sequences within the gray boxes indicate the local contexts in which OG was oxidized.

Figure 3

Context effects on OG oxidation product distributions observed with the oxidants Na₂IrCl₆ (Ir(IV)) and ONOOCO₂⁻ (SIN-1). Each reaction was conducted as described in the text to give ~75% for Na₂IrCl₆ reactions, and ~10% for ONOOCO₂⁻ reactions. Product yields were estimated by integration of peak areas that were normalized by their molar extinction coefficients, and triplicate trials were conducted to obtain suitable errors.

Figure 4

pH Dependency in Sp and Gh yield within each structural context studied. The OG-structural context studied include nucleoside OG (black circle), OGTP (red square), ssOG (aqua triangle), OG•A (dark green square), and OG•C (blue diamond). Solid lines represent fitting to the Henderson-Hasselbalch equation. Error in the data ~5%.

Figure 5

The effect of sequence context on OG oxidation product distributions in each ODN context studied.

Figure 6

Proposed base pairing schemes for the OG•^4EtC and OG•D base pairs. These modified base pairs are compared to their native OG base pairs with respect to structure and relative T_m.

Figure 7

Hydrogen bonding effect on OG oxidation product distributions.

Figure 8

ODN sequences studied to evaluate the context effect on the relative reactivity of OG toward oxidation. The gray highlighted regions show the sequence context in which the OG resides.

Scheme 1

Guanine oxidation pathways.

Scheme 2

Proposed mechanism for pH-dependent of OG oxidation to yield hydantoins Sp and Gh.

Scheme 3

Proposed mechanism for modulated hydantoin formation when OG is oxidized in the OG•C vs. OG•A base pair contexts.