Identification by UV resonance Raman spectroscopy of an imino tautomer of 5-hydroxy-2′-deoxycytidine, a powerful base analog transition mutagen with a much higher unfavored tautomer frequency than that of the natural residue 2′-deoxycytidine

Abstract

UV resonance Raman spectroscopy was used to detect and estimate the frequency of the unfavored imino tautomer of the transition mutagen 5-hydroxy-2-deoxycytidine (HO 5 dCyt) in its anionic form. In DNA, this 2-deoxycytidine analog arises from the oxidation of 2-deoxycytidine and induces C 3 T transitions with 10 2 greater frequency than such spontaneous transitions. An imino tautomer marker carbonyl band (Ϸ1650 cm ؊1 ) is enhanced at Ϸ65°C against an otherwise stable spectrum of bands associated with the favored amino tautomer. This band is similarly present in the UV resonance Raman spectra of the imino cytidine analogs N 3 -methylcytidine at high pH and N 4 -methoxy-2-deoxycytidine at pH 7 and displays features attributable to the imino form of C residues and their derivatives. The fact that the imino tautomer of HO 5 dCyt occurs at a frequency consistent with its high mutagenic enhancement lends strong support to the hypothesis that unfavored base tautomers play important roles in the mispair intermediates of replication leading to substitution mutations.The tautomerism of nucleic acid bases is of great interest because of the putative role of their unfavored tautomers in substitution mutagenesis (1, 2). Thus, whereas the dominant amino and keto forms of the base residues enable correct gene replication by complementary base pairing between A and T and between G and C, the minor tautomeric imino and enol species allow for mispairing of A with C, G with T, and also are involved in mispairing of A with A, G with G, A with G, and G with A, resulting in transition and transversion mutations, respectively.Experimental and theoretical investigations involving pK measurement of various tautomer analogs (3), relaxation methods (4), and quantum mechanical calculations (5) have afforded estimates of the frequency of these rare tautomers. However, because of the very low level of minor tautomeric species in solution (Ϸ10 Ϫ4 -10 Ϫ5 ; refs. 6-8), it has not been possible to observe them directly by spectroscopy. Here, we report evidence obtained by UV resonance Raman spectroscopy (UVRR) for the minor tautomer of a highly mutagenic analog of the 2Ј-deoxycytidine (dCyt) residue, 5-hydroxy-2Ј-deoxycytidine (HO 5 dCyt).Earlier, it was found that C 3 T mutations resulting from transition metal-mediated damage to DNA by reactive oxygen species most frequently occur opposite C residues (9). The major mutagenic modification of these C residues is HO 5 dCyt, an oxidation product. HO 5 dCTP is incorporated efficiently into DNA by a DNA polymerase in vitro (10). On transfection of this DNA into Escherichia coli, it causes C 3 T transitions at a frequency of Ϸ2.5%, very much higher than that observed for any previously identified base analog-induced DNA lesion. A possible mechanism for these transitions involves a much greater frequency of the imino tautomer of HO 5 dCyt, which would increase the tendency to pair with A (2).In fact, our results show that the frequency of the minor tautomer of the anionic form of HO 5 dCyt, wh...

Two-dimensional IR spectroscopy of the anti-HIV agent KP1212 reveals protonated and neutral tautomers that influence pH-dependent mutagenicity

“…This pair of peaks could be attributed to either KI or KA-N 3 . The C=O frequency of N3-methylcytidine, which adopts the ketoimino form, is blue-shifted to ∼1,670 cm −1 (19). Combining this observation with our DFT calculations, which predict a larger peak splitting for KI than for KA-N 3 , we conclude that the 1,675-cm −1 peak corresponds to the C=O stretch of the KI tautomer.…”

“…2 B-E). The pK a1 at 7.0 corresponds to protonation of KP1212, and is significantly higher than that of dC (pK a = 4.3 at N3 atom) (19). This value could be even higher as pK a s of some nucleosides have been found to increase when converted to nucleotides (23).…”

“…Therefore, NMR-a method with a millisecond time resolution-cannot separate short-lived species and offers only an exchange-averaged characterization of the structure (17). By contrast, vibrational spectroscopy is more promising due to its structural sensitivity and picosecond time resolution (18,19). For example, Raman spectroscopy of 5-OH-dC (also a G-to-A mutagen) was used to identify a <1% population of the anionic imino-keto tautomer at high pH, which was proposed to base pair with adenine (19).…”

“…By contrast, vibrational spectroscopy is more promising due to its structural sensitivity and picosecond time resolution (18,19). For example, Raman spectroscopy of 5-OH-dC (also a G-to-A mutagen) was used to identify a <1% population of the anionic imino-keto tautomer at high pH, which was proposed to base pair with adenine (19). Nevertheless, even when using vibrational spectroscopy, tautomers remain difficult to separate due to peak overlap and uncertainty in peak assignments.…”

Direct observation of ground-state lactam–lactim tautomerization using temperature-jump transient 2D IR spectroscopy

“…To characterize the tautomeric equilibria, researchers have used techniques such as UV absorption (4), circular dichroism (5), X-ray crystallography (3), NMR (6), Raman (7), and IR absorption spectroscopy (8). However, these methods face several challenges to characterizing thermodynamic and kinetic data for tautomeric equilibria and exchange processes.…”