Development of enzymatic probes of oxidative and nitrosative DNA damage caused by reactive nitrogen species

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Abstract

Chronic inflammation is associated with a variety of human diseases, including cancer, with one possible mechanistic link involving over-production of nitric oxide (NOradical dot) by activated macrophages. Subsequent reaction of NOradical dot with superoxide in the presence of carbon dioxide yields nitrosoperoxycarbonate (ONOOCO2), a strong oxidant that reacts with guanine in DNA to form a variety of oxidation and nitration products, such 2′-deoxy-8-oxoguanosine. Alternatively, the reaction of NOradical dot and O2 leads to the formation of N2O3, a nitrosating agent that causes nucleobase deamination to form 2′-deoxyxanthosine (dX) and 2′-deoxyoxanosine (dO) from dG; 2′-deoxyinosine (dI) from dA; and 2′-deoxyuridine (dU) from dC, in addition to abasic sites and dG–dG cross-links. The presence of both ONOOCO2 and N2O3 at sites of inflammation necessitates definition of the relative roles of oxidative and nitrosative DNA damage in the genetic toxicology of inflammation. To this end, we sought to develop enzymatic probes for oxidative and nitrosative DNA lesions as a means to quantify the two types of DNA damage in in vitro DNA damage assays, such as the comet assay and as a means to differentially map the lesions in genomic DNA by the technique of ligation-mediated PCR. On the basis of fragmentary reports in the literature, we first systematically assessed the recognition of dX and dI by a battery of DNA repair enzymes. Members of the alkylpurine DNA glycosylase family (E. coli AlkA, murine Aag, and human MPG) all showed repair activity with dX (kcat/Km 29 × 10−6, 21 × 10−6, and 7.8 × 10−6 nM−1 min−1, respectively), though the activity was considerably lower than that of EndoV (8 × 10−3 nM−1 min−1). Based on these results and other published studies, we focused the development of enzymatic probes on two groups of enzymes, one with activity against oxidative damage (formamidopyrimidine-DNA glycosylase (Fpg); endonuclease III (EndoIII)) and the other with activity against nucleobase deamination products (uracil DNA glycosylase (Udg); AlkA). These combinations were assessed for recognition of DNA damage caused by N2O3 (generated with a NOradical dot/O2 delivery system) or ONOOCO2 using a plasmid nicking assay and by LC–MS analysis. Collectively, the results indicate that a combination of AlkA and Udg react selectively with DNA containing only nitrosative damage, while Fpg and EndoIII react selectively with DNA containing oxidative base lesions caused by ONOOCO2. The results suggest that these enzyme combinations can be used as probes to define the location and quantity of the oxidative and nitrosative DNA lesions produced by chemical mediators of inflammation in systems, such as the comet assay, ligation-mediated polymerase chain reaction, and other assays of DNA damage and repair.

Introduction

DNA repair enzymes have frequently been employed as probes of DNA damage in a variety of applications (e.g., Refs. [1], [2]). In most instances, the enzymes are used to convert classes of nucleobase lesions to single-strand breaks, or to abasic sites that could be converted to single-strand breaks, followed by quantification of the breaks using techniques, such as alkaline elution [3], alkaline unwinding [4], single-cell gel electrophoresis (comet assay) [5], [6], [7], [8], nick translation [9], and the plasmid nicking assay [1], [2], [10], [11]. The base lesions and strand breaks can also be mapped to single-nucleotide resolution in cells by means of ligation-mediated PCR (LMPCR; e.g., Refs. [12], [13]). The use of combinations of repair enzymes provides a means to distinguish different classes of DNA damage that arise by different chemical mechanisms, thus providing evidence to identify the species responsible for the damage. Several DNA repair enzymes have been developed as selective probes for oxidized purines (Fpg; Refs. [14], [15]), oxidized pyrimidines (EndoIII; Ref. [16]), and bulky DNA adducts, such as benzo[a]pyrene (uvrABC; Ref. [17]), among others. However, comparable probes for products of nitrosative deamination of DNA bases have not been assessed. These DNA lesions are associated with nitric oxide (NOradical dot) overproduction in states of inflammation and are the focus of the present studies.

Chronic inflammation has long been recognized as a risk factor for the development of a variety of human cancers [18], [19]. One possible chemical link between inflammation and human disease involves unregulated production of NOradical dot by macrophages, which subsequently leads to high levels of derivative reactive nitrogen species that may be involved in pathological processes. In particular, macrophages activated as part of the inflammatory process produce large quantities of both NOradical dot and superoxide (O2radical dot) that react to form peroxynitrite (ONOO) and ultimately, in the presence of carbon dioxide, nitrosoperoxycarbonate (ONOOCO2). The latter is a strongly oxidizing species that selectively oxidizes guanine bases in DNA to form 8-nitro-2′-deoxyguanosine (8-nitro-dG), 8-oxo-2′-deoxyguanosine (8-oxo-dG), and at biologically relevant ONOOCO2 concentrations, several of the nitration and oxidation products shown in Fig. 1 [20]. NOradical dot can also react with molecular oxygen (O2) to form nitrous anhydride (N2O3), a powerful nitrosating agent capable of causing nucleobase deamination in DNA [20]. In addition to dG–dG cross-links, N2O3 causes nitrosative deamination of dG to produce 2′-deoxyxanthosine (dX) and 2′-deoxyoxanosine (dO); of dC to produce 2′-deoxyuridine (dU); and of dA to yield 2′-deoxyinosine (dI) (Fig. 1) [20], [21]. It is important to point out that both the oxidative and nitrosative DNA lesions associated with inflammation are also produced by other genotoxic agents. For example, the deamination of nucleobases in DNA and RNA can occur by simple hydrolysis, as well as by specific RNA and DNA deaminases [22], [23], [24], [25]. This highlights the broad utility of enzymatic probes of oxidative and nitrosative DNA damage.

While sensitive analytical methods have been developed for the DNA lesions associated with inflammation [20], [21], there is a demand for alternative approaches to quantifying and localizing the DNA lesions in applications, such as LMPCR and the comet assay. To this end, we undertook the development of DNA glycosylases as probes of oxidative and nitrosative DNA damage in DNA exposed to ONOOCO2 and N2O3. In this study, supercoiled plasmid pUC19 DNA was first treated with ONOOCO2 by bolus addition of ONOO to DNA in a bicarbonate-containing solution, or with N2O3 in the Silastic tubing-based NOradical dot/O2 delivery system [21]. DNA damage profiles induced by ONOOCO2 and N2O3 were obtained after treatment with each group of enzymes and validated by LC–MS analysis [21]. Our results indicate that E. coli 3-methyladenine-DNA glycosylase II (AlkA), endonuclease V (Endo V) and uracil DNA glycosylase (Udg) react selectively with DNA exposed to N2O3, while Fpg and endonuclease III (EndoIII) react selectively with DNA treated with ONOOCO2.

Section snippets

Materials

All chemicals and reagents were of the highest purity available and were used without further purification unless noted otherwise. ONOO was synthesized by ozonolysis of azide as described by Pryor et al. [26] and was characterized spectrophotometrically in 0.1N NaOH (ɛ302 = 1670 M−1 cm−1) and for its ability to cause the nitration of l-tyrosine using a method described by Beckman et al. [27] and modified by Pryor et al. [26]. [γ-32P]ATP was obtained from NEN Biosciences (Piscataway, NJ). Agarose

Recognition of dX and dI by DNA repair enzymes

Several groups have identified repair enzymes that recognize products of nitrosative DNA damage [38], [39], [40], [41], [42]. The fragmentary nature of these reports motivated us to undertake a survey of the activities of a variety of DNA repair enzymes with substrates containing dX and dI. An initial assessment of the recognition of dX-containing substrates was performed by treating oligodeoxynucleotides with the repair enzymes followed by sequencing gel resolution of the cleavage products, as

Discussion

There is growing interest in the formation and repair of DNA lesions caused by the reactive nitrogen species arising at sites of inflammation, with a recognition of two major DNA damage chemistries: oxidation and nitrosation [20]. With the goal of distinguishing between these two chemistries in terms of DNA lesions, we sought to develop enzymatic tools to localize and quantify oxidative and nitrosative DNA lesions in isolated DNA and cells exposed to reactive nitrogen species. Such tools would

Conclusions

We have performed quantitative studies to identify DNA repair enzymes that distinguish between the two major forms of DNA damage chemistry, oxidation, and nitrosation, caused by reactive nitrogen species arising at sites of inflammation. The results revealed that Fpg and EndoIII selectively recognize ONOOCO2-induced DNA damage but had little reactivity with N2O3-treated DNA, while the opposite was true for a combination of AlkA and Udg. Though the ability to differentiate the different damage

Acknowledgments

The authors wish to thank Prof. Sankar Mitra (University of Texas Medical Branch) for providing human endonuclease III, Prof. Bruce Demple (Harvard School of Public Health) for human AP endonuclease, Prof. Thomas Ellenberger (Harvard Medical School) for providing AlkA, and Drs. Koli Taghizadeh and John Wishnok for assistance with the LC/MS analyses performed in the MIT Center for Environmental Health Sciences Bioanalytical Facilities Core. Financial support for this work was provided by

References (86)

  • W.A. Pryor et al.

    A practical method for preparing peroxynitrite solutions of low ionic strength and free of hydrogen peroxide

    Free Rad. Biol. Med.

    (1995)
  • J.S. Beckman et al.

    Kinetics of superoxide dismutase- and iron-catalyzed nitration of phenolics by peroxynitrite

    Arch. Biochem. Biophys.

    (1992)
  • D.M. Wilson et al.

    Incision activity of human apurinic endonuclease (Ape) at abasic site analogs in DNA

    J. Biol. Chem.

    (1995)
  • M. Yao et al.

    Further characterization of Escherichia coli endonuclease V. Mechanism of recognition for deoxyinosine, deoxyuridine, and base mismatches in DNA

    J. Biol. Chem.

    (1997)
  • S. Ikeda et al.

    Purification and characterization of human NTH1, a homolog of Escherichia coli endonuclease III. Direct identification of Lys-212 as the active nucleophilic residue

    J. Biol. Chem.

    (1998)
  • J.S. Beckman et al.

    Reactions of nitric oxide, superoxide and peroxynitrite with superoxide dismutase in neurodegeneration

    Prog. Brain Res.

    (1994)
  • B. He et al.

    Deoxyxanthosine in DNA is repaired by Escherichia coli endonuclease V

    Mutat. Res.

    (2000)
  • H. Kamiya et al.

    Probing the substrate recognition mechanism of the human MTH1 protein by nucleotide analogs

    J. Mol. Biol.

    (2004)
  • Y.W. Kow

    Repair of deaminated bases in DNA

    Free Radic. Biol. Med.

    (2002)
  • K.A. Schouten et al.

    Endonuclease V protects Escherichia coli against specific mutations caused by nitrous acid

    Mutat. Res.

    (1999)
  • N.Y. Tretyakova et al.

    Peroxynitrite-induced DNA damage in the supF gene: correlation with the mutational spectrum

    Mutat. Res.

    (2000)
  • D. Chakravarti et al.

    Cloning and expression in Escherichia coli of a human cDNA encoding the DNA repair protein N-methylpurine-DNA glycosylase

    J. Biol. Chem.

    (1991)
  • L. Xia et al.

    Human 3-methyladenine-DNA glycosylase: effect of sequence context on excision association with PCNA, and stimulation by AP endonuclease

    J. Mol. Biol.

    (2005)
  • S. Bjelland et al.

    DNA glycosylase activities for thymine residues oxidized in the methyl group are functions of the AlkA enzyme in Escherichia coli

    J. Biol. Chem.

    (1994)
  • J. Tuo et al.

    Importance of guanine nitration and hydroxylation in DNA in vitro and in vivo

    Free Radic. Biol. Med.

    (2000)
  • V. Yermilov et al.

    Formation of 8-nitroguanine in DNA treated with peroxynitrite in vitro and its rapid removal from DNA by depurination

    FEBS Lett.

    (1995)
  • E.I. Zaika et al.

    Substrate discrimination by formamidopyrimidine-DNA glycosylase: a mutational analysis

    J. Biol. Chem.

    (2004)
  • D. Gasparutto et al.

    Repair and replication of oxidized DNA bases using modified oligodeoxyribonucleotides

    Biochimie

    (2000)
  • Z. Hatahet et al.

    New substrates for old enzymes. 5-Hydroxy-2′-deoxycytidine and 5-hydroxy-2′-deoxyuridine are substrates for Escherichia coli endonuclease III and formamidopyrimidine DNA N-glycosylase, while 5-hydroxy-2′-deoxyuridine is a substrate for uracil DNA N-glycosylase

    J. Biol. Chem.

    (1994)
  • A.A. Purmal et al.

    Enzymatic processing of uracil glycol, a major oxidative product of DNA cytosine

    J. Biol. Chem.

    (1998)
  • S.S. Wallace

    Biological consequences of free radical-damaged DNA bases

    Free Radic. Biol. Med.

    (2002)
  • B. Epe et al.

    DNA damage by peroxynitrite characterized with DNA repair enzymes

    Nucleic Acids Res.

    (1996)
  • G.K. Kuipers et al.

    Characterization of DNA damage induced by gamma-radiation-derived water radicals, using DNA repair enzymes

    Int. J. Radiat. Biol.

    (1998)
  • A.R. Collins

    The comet assay for DNA damage and repair: principles, applications, and limitations

    Mol. Biotechnol.

    (2004)
  • J.P. Pouget et al.

    DNA damage induced in cells by gamma and UVA radiation as measured by HPLC/GC–MS and HPLC–EC and Comet assay

    Chem. Res. Toxicol.

    (2000)
  • C. Collins et al.

    Analysis of 3′-phosphoglycolaldehyde residues in oxidized DNA by gas chromatography/negative chemical ionization/mass spectrometry

    Chem. Res. Toxicol.

    (2003)
  • L.H. Breimer et al.

    Thymine lesions produced by ionizing radiation in double-stranded DNA

    Biochemistry

    (1985)
  • S. Tornaletti et al.

    Slow repair of pyrimidine dimers at p53 mutation hotspots in skin cancer

    Science

    (1994)
  • J. Xu et al.

    DNA damage produced by enediynes in the human phosphoglycerate kinase gene in vivo: esperamicin A1 as a nucleosome footprinting agent

    Biochemistry

    (1998)
  • S. Boiteux et al.

    Substrate specificity of the Escherichia coli Fpg protein (formamidopyrimidine-DNA glycosylase): excision of purine lesions in DNA produced by ionizing radiation or photosensitization

    Biochemistry

    (1992)
  • M.F. Denissenko et al.

    Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in P53

    Science

    (1996)
  • G.H. Cassell

    Infectious causes of chronic inflammatory diseases and cancer

    Emerg. Infect. Dis.

    (1998)
  • M. Dong et al.

    Absence of 2′-deoxyoxanosine and presence of abasic sites in DNA exposed to nitric oxide at controlled physiological concentrations

    Chem. Res. Toxicol.

    (2003)
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