Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
Development of enzymatic probes of oxidative and nitrosative DNA damage caused by reactive nitrogen species
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 (NO) 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 NO 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 NO and superoxide (O2) 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]. NO 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 NO/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
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2010, Journal of Biological ChemistryCitation Excerpt :This result indicated that the E. coli genome contained an additional XDG enzyme. Given that a previous study had investigated XDG activity in purified E. coli AlkA, endo III, endo V, endo VIII, Fpg/MutM, MutY, and UDG (11), we surmised that MUG, which was not included in the previous study, may be accountable for the observed XDG activity in the triple mutant cell extract. A quick test was performed using purified MUG from a commercial source (Trevigen).
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2009, Journal of Molecular BiologyCitation Excerpt :In an earlier study, repair of xanthine lesions was observed in human lymphoblast cells.36 Later, it was found that human alkyladenine DNA glycosylase (hAAG) exhibited strong XDG activity12,32 (L.D. and W.C., unpublished data). hAAG is active toward xanthine in both ssDNA and dsDNA regardless of the opposite base (L.D. and W.C., unpublished data).
- 1
Present address: Novartis Institutes for BioMedical Research, Models of Disease Center, 250 Massachusetts Ave., Cambridge, MA 02139, USA.
- 2
Present address: ArQule, 19 Presidential Way, Woburn, MA 01801, USA.
- 3
Present address: Université Laval, Quebec City, QC, Canada G1K 7P4.