8-hydroxyguanosine and spiroiminodihydantoin

Damage to the genome is implicated in the progression of cancer and stress-induced diseases. DNA lesions exist in low levels, and cannot be amplified by standard PCR because they are frequently strong blocks to polymerases. Here, we describe a method for PCR amplification of lesion-containing DNA in which the site and identity could be marked, copied and sequenced. Critical for this method is installation of either the dNaM or d5SICS nucleotides at the lesion site after processing via the base excision repair process. These marker nucleotides constitute an unnatural base pair, allowing large quantities of marked DNA to be made by PCR amplification. Sanger sequencing confirms the potential for this method to locate lesions by marking, amplifying and sequencing a lesion in the KRAS gene. Detection using the α-hemolysin nanopore is also developed to analyse the markers in individual DNA strands with the potential to identify multiple lesions per strand.

Topics: Base Pairing; DNA; DNA Damage; Genes, ras; Guanosine; Nanopores; Nucleotides; Polymerase Chain Reaction; Sequence Analysis, DNA; Spiro Compounds; Uracil; Vascular Endothelial Growth Factor A

8-Oxo-7,8-dihydro-2'-deoxyguanosine (OG), a prevalent product of oxidative stress on cellular DNA, is readily further oxidized forming adducts with nucleophiles. In the presence of tyrosine or p-cresol, an unusual tricyclo[4.3.3.0] adduct has been characterized in both nucleoside and oligodeoxynucleotide studies. The adduct is more stable in oligomers than nucleosides and undergoes slow reversion and hydration to spiroiminodihydantoin.

Topics: DNA; DNA Adducts; DNA Damage; Guanosine; Oligodeoxyribonucleotides; Oxidative Stress; Proteins; Spiro Compounds; Tyrosine

Base excision repair (BER) is the major pathway employed to excise oxidized DNA lesions. Human Neil1, a versatile glycosylase in the BER pathway, repairs a diverse array of oxidative lesions; however, the most prevalent, 8-oxo-7,8-dihydroguanine (8-oxoG), is only weakly excised. The structural origin of hNeil1's ability to repair a variety of lesions but not 8-oxoG is a model system for connecting enzyme structure and lesion-recognition specificity. To elucidate structural properties determining hNeil1's substrate specificities, we have investigated it in complex with two pairs of representative well-repaired substrates: the R- and S-spiroiminodihydantoin (Sp) stereoisomers, nonplanar further oxidation products of guanine, and the 5R,6S- and 5S,6R-thymine glycol (Tg) stereoisomers, the most prevalent oxidative lesions of thymine. We also investigate the poorly repaired 8-oxoG. We employed molecular modeling and 10 ns molecular dynamics (MD) simulations. The results of our investigations provide structural explanations for the ability of hNeil1 to excise a variety of oxidative lesions: they possess common chemical features, namely, a pyrimidine-like ring and shared hydrogen bond donor-acceptor properties, which allow the lesions to fit well in the binding pocket, which is somewhat flexible. However, the planar 8-oxoG is not as well accommodated in the shallow and comparatively cramped recognition pocket; it has fewer hydrogen bonding interactions with the enzyme and a solvent exposed six-membered ring, consistent with its poor repair susceptibility by this enzyme.

Topics: Computer Simulation; DNA Glycosylases; DNA Repair; DNA-Binding Proteins; Guanosine; Humans; Models, Molecular; Oxidative Stress; Protein Binding; Pyrimidines; Spiro Compounds; Static Electricity; Stereoisomerism; Substrate Specificity; Thermodynamics

8-Oxoguanine (8-oxoG) is an unstable mutagenic DNA lesion that is prone to further oxidation. High valent metals such as Cr(V) and Ir(IV) readily oxidize 8-oxoG to form guanidinohydantoin (Gh), its isomer iminoallantoin (Ia), and spiroiminodihydantoin (Sp). When present in DNA, these lesions show enhanced base misincorporation over the parent 8-oxoG lesion leading to G --> T and G --> C transversion mutations and polymerase arrest. These findings suggested that further oxidized lesions of 8-oxoG are more mutagenic and toxic than 8-oxoG itself. Repair of oxidatively damaged bases, including Sp and Gh/Ia, are initiated by the base excision repair (BER) system that involves the DNA glycosylases Fpg, Nei, and Nth in E. coli. Mammalian homologs of two of these BER enzymes, OGG1 and NTH1, have little or no affinity for Gh/Ia and Sp. Herein we report that two recently identified mammalian glycosylases, NEIL1 and NEIL2, showed a high affinity for recognition and cleavage of DNA containing Gh/Ia and Sp lesions. NEIL1 and NEIL2 recognized both of these lesions in single-stranded DNA and catalyzed the removal of the lesions through a beta- and delta-elimination mechanism. NEIL1 and NEIL2 also recognized and excised the Gh/Ia lesion opposite all four natural bases in double-stranded DNA. NEIL1 was able to excise the Sp lesion opposite the four natural bases in double-stranded DNA, however, NEIL2 showed little cleavage activity against the Sp lesion in duplex DNA although DNA trapping studies show recognition and binding of NEIL2 to this lesion. This work suggests that NEIL1 and NEIL2 are essential in the recognition of further oxidized lesions arising from 8-oxoG and implies that these BER glycosylases may play an important role in the repair of DNA damage induced by carcinogenic metals.

Topics: Animals; Chromatography, High Pressure Liquid; Chromium Compounds; Cloning, Molecular; DNA Damage; DNA Glycosylases; DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase; Guanidines; Guanosine; Hydantoins; Mice; Oligonucleotides; Spiro Compounds

Peroxynitrite-mediated oxidation of 8-oxoguanosine results in the formation of two product classes distinguished by the source of their incorporated oxygen atoms. The first product class consists of dehydroguanidinohydantoin (DGh), N-nitro-dehydroguanidinohydantoin (NO2-DGh), and 2,4,6-trioxo[1,3,5]triazinane-1-carboxamidine (CAC) with peroxynitrite as the exogenous O atom source, and the second includes spiroiminodihydantoin (Sp), guanidinohydantoin (Gh), and 4-hydroxy-2,5-dioxo-imidazolidine-4-carboxylic acid (HICA), with water serving as the exogenous O atom source. The first product class forms exclusively at high peroxynitrite fluxes, while the second forms exclusively at limiting peroxynitrite fluxes. At intermediate peroxynitrite fluxes, both sets of products are formed. At high fluxes, DGh was the major reaction product, and after several of the peroxynitrite-derived radicals were eliminated as the exogenous O atom source, the peroxynitrite anion emerged as the most likely candidate. On the other hand, at lower fluxes, either Gh or Sp was the major product, depending on the pH of the reaction mixture. At low and high pH, respectively, Gh and Sp were the major products, and the plot of pH vs ratio of Sp/(Sp+Gh) had an inflection at pH 5.8. Interestingly, the pH dependence for oxidation of 8-oxoGuo with CoCl2 and KHSO5 was identical to that for oxidation by peroxynitrite, indicating that the phenomenon arises due to characteristics of an 8-oxoGuo-derived rather than an oxidant-derived intermediate, since these two systems generate different reactive species. On the basis of these findings, a model in which 8-oxoGuo is oxidized to the bisimine intermediate, 1 is proposed. At high peroxynitrite fluxes, the reaction of 1 with ONOO- predominates over the reaction with H2O, leading exclusively to DGh, NO2-DGh, and CAC, while at limiting peroxynitrite concentrations, the reaction with H2O dominates, and Gh and Sp are formed exclusively. At intermediate peroxynitrite fluxes, the relative kinetics of the reaction between 1 and ONOO- or H2O are such that both product classes are formed. To explain the pH-dependent Gh and Sp yields, we propose that 5 has a pKa approximately 5.8 and that the differential reactivity of the protonated and deprotonated form of 5 leads to its partitioning into Gh and Sp, respectively.

Topics: Chromatography, High Pressure Liquid; Guanidines; Guanosine; Models, Chemical; Nitrates; Oxidation-Reduction; Oxygen Isotopes; Spiro Compounds

The photosensitized oxidation of 2',3',5'-tris-(O-tert-butyldimethylsilyl)-8-oxo-7,8-dihydroguanosine (8-oxoG) with singlet oxygen was studied by low-temperature NMR. A stable intermediate was characterized at -60 degrees C by (13)C, 2D NMR HMBC spectra, and chemical shifts calculated by hybrid Hartree-Fock density functional theory which agreed with the structure 5-hydroperoxy-8-oxo-7,8-dihydroguanosine. Reduction of this intermediate at low temperature afforded the corresponding alcohol, the long-postulated 5-hydroxy-8-oxo-7,8-dihydroguanosine, the last intermediate in the formation of spiroiminodihydantoin. Upon warming to room temperature, this alcohol rearranges to form the spiroiminodihydantoin in good yield within 2 h.

Topics: Cold Temperature; Guanosine; Kinetics; Nuclear Magnetic Resonance, Biomolecular; Oxidation-Reduction; Photochemistry; Spiro Compounds

[reaction: see text]. The potent oxidant, peroxynitrite, will oxidize 8-oxo-7,8-dihydroguanosine to give several products. In the presence of a thiol agent, the major final product has been determined to be a spiroiminodihydantoin compound. Additionally, we have found that the spiroiminodihydantoin, and not the previously reported 4-hydroxy-8-oxo-4,8-dihydroguanosine, is the major final product formed during the methylene blue-mediated photooxidation of guanosine.

Topics: Chromatography, High Pressure Liquid; Guanosine; Methylene Blue; Molecular Structure; Nitrates; Oxidants; Oxidation-Reduction; Spiro Compounds; Sulfhydryl Compounds

[reaction: see text] Further oxidation of the common DNA lesion 8-oxo-7,8-dihydroguanosine by one-electron oxidants such as IrCl6(2-), Fe(CN)6(3-), or SO4-* leads to two major products, depending upon reaction conditions. In nucleosides at pH 7, 22 degrees C, the principal product is shown herein to be a spiroiminodihydantoin nucleoside, as a diastereomeric mixture, that can be characterized by NMR, ESI-MS/MS, and independent synthesis.

Topics: Crystallography, X-Ray; Electrons; Guanosine; Magnetic Resonance Spectroscopy; Mass Spectrometry; Oxidation-Reduction; Spiro Compounds