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 the cell where it resides in many structural contexts including the nucleotide pool, single-stranded DNA at transcription forks and replication bubbles, and in duplex DNA base paired with either A or 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, N4-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 pKa 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.

Base and Nucleotide Excision Repair of Oxidatively Generated Guanine Lesions in DNA

“…The oligonucleotide 5Ј-CCATC(Sp)CTACC adduct containing the Sp-S diastereomer (Sp1) was the first-eluting fraction, whereas oligonucleotides with the Sp-R diastereomer (Sp2) eluted in the second fraction on a DNAPac PA-100 column (Dionex, Sunnyvale, CA) (24). The Gh-modified oligonucleotides were prepared by oxidation of the oligonucleotide 5Ј-CCATC(8-oxoG)-CTACC adducts with (NH 4 ) 2 IrCl 6 complex at pH 6.0 (12,21). The oligonucleotides with single NIm lesions were generated by oxidation of guanine in the same 5Ј-CCATCGCTACC sequence context using a photochemical method to generate carbonate radical anion/nitrogen dioxide radical species at pH 7.5-8.0 and isolated by reversed-phase HPLC (17,41).…”

8-Oxo-7,8-dihydro-2′-deoxyguanosine and abasic site tandem lesions are oxidation prone yielding hydantoin products that strongly destabilize duplex DNA

“…1). 39 By directly monitoring the oxidation products in the intact strand, we can more confidently quantify the yields, in contrast to nuclease digestion of the strands to nucleosides for analysis that can lead to incomplete digestion impacting the products quantified.…”

“…The hydantoins dGh and dSp are formed by oxidation of dOG via the oxidants HO · , 37 CO 3 ·- , 12,38,39 · NO 2 , 40 Na 2 IrCl 6 , 22,39 K 2 IrBr 6 , 39 K 3 Fe(CN) 6 , 39 CoCl 2 /KHSO 5 , 38 chromate complexes, 26 and photooxidants when H 2 O is the nucleophile. 20,41,42 The dGh lesion dominates in reactions conducted at pH < 5.7, 22,39 or in dsODNs with dOG base paired with dC; 26,39 in contrast, dSp yields dominate in reactions with pH > 5.7, 39,42 in ssODN, 26,39 and in G-quadruplex contexts. 15 The mechanistic details to understand the pH dependency in the hydantoin yields were studied and established by density functional theory (DFT) to identify that a key intermediate leading to dGh or dSp has a titratable site that determines the reaction course.…”

Sequence selective tagging of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) using PNAs

“…8,9 8-OxodGuo is even more readily oxidized than dG and serves as the precursor to a number of other mutagenic lesions. [10][11][12][13][14] Consequently, there is significant interest in using 8-oxodGuo as a biomarker, which requires developing methods for its quantitation in DNA. 15,16 Detecting damaged nucleotides at specific positions within DNA is potentially useful for a variety of reasons, including as an early risk assessment by providing correlations between mutagenic hotspots and the formation/persistence of specific lesions at those same positions.…”

“…Use of either oxidant provided P80% of the tagged product with the matched sequence (9). However, the stronger iridium oxidant yielded P25% of tagged material with the mismatched 8-oxodGuo substrate (10) or the 51 bp DNA lacking the lesion (11). Fortunately, no tagging of 10 or 11 was observed when ferricyanide was employed.…”