pH-Dependent Equilibrium between 5-Guanidinohydantoin and Iminoallantoin Affects Nucleotide Insertion Opposite the DNA Lesion

Abstract

Four-electron oxidation of 2’-deoxyguanosine (dG) yields 5-guanidinohydantoin (dGh) as a product. Previously, we hypothesized that dGh could isomerize to iminoallantoin (dIa) via a mechanism similar to the isomerization of allantoin. The isomerization reaction was monitored by HPLC and found to be pH dependent with a transition pH = 10.1 in which dGh was favored at low pH and dIa was favored at high pH. The structures for these isomers were confirmed by UV-vis, MS, 1H, and 13C NMR. Additionally, the UV-vis and NMR experimental results are supported by density functional theory calculations. A mechanism is proposed to support the pH dependency of the isomerization reaction. Next, we noted the hydantoin ring of dGh mimics thymine, while the iminohydantoin ring of dIa mimics cytosine; consequently, a dGh/dIa site was synthesized in a DNA template strand and standing start primer extension studies were conducted with Klenow fragment exo−. The dATP/dGTP insertion ratio opposite the dGh/dIa site as a function of pH was evaluated from pH 6.5 – 9.0. At pH 6.5 only dATP was inserted, but as the pH increased to 9.0, the amount of dGTP insertion steadily increased. This observation supports dGh to dIa isomerization in DNA with a transition pH of ~8.2.

Formation and processing of DNA damage substrates for the hNEIL enzymes

“…The hydantoins can also form directly from G (Scheme 1), as described below. Recently, our laboratory demonstrated that Gh is in a pH-dependent equilibrium with its constitutional isomer iminoallantoin (Ia, Scheme 1) [56], supporting a past hypothesis of ours [32]. Lastly, six-electron oxidation of G yields oxidized 5-guanidinohydantoin (Gh ox , Scheme 1) that has limited stability and decomposes through several intermediates to yield a 2′-deoxyribosyl-urea lesion [16].…”

“…Furthermore, Sp, Gh, Ia, and 2Ih nucleosides are generated as pairs of diastereomers, and each of them elutes from an HPLC column as two peaks [33, 56–60]. Additionally, the pH-dependent isomerization between Gh and Ia dramatically impacts the elution time of these isomers and must be considered during product analysis [56].…”

Removal of oxidatively generated DNA damage by overlapping repair pathways

“…2) [55]. In DNA, the isomerization of Gh to Ia occurs in basic solutions (pH > 8.2) [64]. The Sp and Gh lesions are products of four-electron oxidation mechanisms of guanine (in green color in Fig.…”

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

“…The elution profile for dOG, dGh, and dSp on this column was previously established. 44,45 The denaturing HPLC was conducted on a C18 reversed-phase HPLC column with the mobile phases A = 100 mM TEAA pH 7 and 1 M urea in ddH 2 O, and B = 1:1 CH 3 CN:ddH 2 O with 100 mM TEAA and 1 M urea and a flow rate of 1 mL/min with the column heated to 65 °C while monitoring the elution profile by the UV absorbance at 260 nm. The method consisted of starting at 10% B followed by a linear increase to 65% B over 60 min.…”

“…43 The hydantoins dGh and dSp are each diastereotopic. The dGh diastereomers readily interconvert and can further isomerize to the constitutional isomer iminoallantoin-2′-deoxyribose (dIa) at pH > 8.2 in DNA (Scheme 1), 20,44 while the dSp diastereomers are stable, readily resolved by HPLC, and the absolute configurations for them are known. 45–47 These previous works provide background knowledge for the current studies.…”