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Öğe Arabidopsis thaliana Ogg1 protein excises 8-hydroxyguanine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine from oxidatively damaged DNA containing multiple lesions(Amer Chemical Soc, 2003) Morales-Ruiz, T; Birincioglu, M; Jaruga, P; Rodriguez, H; Roldan-Arjona, T; Dizdaroglu, MA functional homologue of eukaryotic Ogg1 proteins in the model plant Arabidopsis thaliana has recently been cloned, isolated, and characterized [Garcia-Ortiz, M. V., Ariza, R. R., and RoldanA Arjona, T. (2001) Plant Mol. Biol. 47,795-804]. This enzyme (AtOgg1) exhibits a high degree of sequence similarity in several highly conserved regions with Saccharomyces cerevisiae, Drosophila melanogaster, and human Ogg1 proteins. We investigated the substrate specificity and kinetics of AtOgg1 for excision of modified bases from oxidatively damaged DNA that contained multiple pyrimidine- and purine-derived lesions. Two different DNA substrates prepared by exposure to ionizing radiation in aqueous solution under N2O or air were used for this purpose. Gas chromatography/isotope-dilution mass spectrometry was applied to identify and quantify modified bases in DNA samples. Of the 17 modified bases identified in DNA samples, only 8-hydroxyguanine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine were significantly excised from both DNA substrates. This is in agreement with the substrate specificities of other eukaryotic Ogg1 proteins that had previously been studied under identical conditions. Excision depended on incubation time, enzyme concentration, and substrate concentration and followed Michaelis-Menten kinetics. A significant dependence of excision on the nature of DNA substrate was observed in accord with previous studies on other DNA glycosylases. A comparison of excision kinetics pointed to significant differences between AtOgg1 and other Ogg1 proteins. We also investigated the effect of base-pairing on the excision using double-stranded oligodeoxynucleotides that contained 8-OH-Gua paired with each of the four DNA bases. The activity of AtOgg1 was most effective on the 8-OH-Gua:C pair with some or very low activity on other pairs in agreement with the activity of other Ogg1 proteins. The results unequivocally show that AtOgg1 possesses common substrates with other eukaryotic Ogg1 proteins albeit significant differences between their excision kinetics.Öğe Biomarkers of oxidative DNA damage used to detect genetic changes in tissue-engineered skin(Pergamon-Elsevier Science Ltd, 2002) Rodriguez, H; Birincioglu, M; Jaruga, P; Barker, PE; O'Connell, C; Dizdaroglu, M[Abstract Not Available]Öğe DNA base damage by the antitumor agent 3-amino-1,2,4-benzotriazine 1,4-dioxide (tirapazamine)(Amer Chemical Soc, 2003) Birincioglu, M; Jaruga, P; Chowdhury, G; Rodriguez, H; Dizdaroglu, M; Gates, KSTirapazamine is a bioreductively activated DNA-damaging agent that selectively kills the hypoxic cells found in solid tumors. This compound shows clinical promise and is currently being examined in a variety of clinical trials, including several phase III studies. It is well established that DNA is an important cellular target for tirapazamine; however, the structural nature of the DNA damage inflicted by this drug remains poorly understood. As part of an effort to understand the chemical events responsible for the hypoxia-selective cytotoxicity of this drug, the studies reported here are designed to characterize tirapazamine-mediated damage to the genetic information stored in the heterocyclic base residues of double-stranded DNA. Here, we used gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry to characterize and quantify oxidative DNA base damage mediated by tirapazamine. A multiplicity of modified bases including 8,5'-cyclopurine-2'-deoxynucleoside tandem lesions were identified and quantified. The results provide the first detailed insight regarding the structural identity of the DNA base lesions caused by this drug. Interestingly, it appears that the hypoxic conditions under which tirapazamine operates, along with the unique chemical properties of the drug, yield a unique variety of DNA base damage that is dominated by formamidopyrimidine and 5-hydroxy-6-hydropyrimidine lesions. Importantly, the results suggest that tirapazamine may generate a set of poorly repaired, potentially cytotoxic DNA base lesions that block DNA transcription and replication. Overall, the results indicate that DNA base damage may contribute to the biological effects of tirapazamine in vivo.Öğe Free radical-induced damage to DNA: Mechanisms and measurement(Elsevier Science Inc, 2002) Dizdaroglu, M; Jaruga, P; Birincioglu, M; Rodriguez, HFree radicals are produced in cells by cellular metabolism and by exogenous agents. These species react with biomolecules in cells, including DNA. The resulting damage to DNA, which is also called oxidative damage to DNA, is implicated in mutagenesis, carcinogenesis, and aging. Mechanisms of damage involve abstractions and addition reactions by free radicals leading to carbon-centered sugar radicals and OH- or H-adduct radicals of heterocyclic bases. Further reactions of these radicals yield numerous products, Various analytical techniques exist for the measurement of oxidative damage to DNA. Techniques that employ gas chromatography (GC) or liquid chromatography (LC) with mass spectrometry (MS) simultaneously measure numerous products, and provide positive identification and accurate quantification. The measurement of multiple products avoids misleading conclusions that might be drawn from the measurement of a single product, because product levels vary depending on reaction conditions and the redox status of cells. In the past, GUMS was used for the measurement of modified sugar and bases, and DNA-protein cross-links. Recently, methodologies using LC/tandem MS (LC/MS/MS) and LC/MS techniques were introduced fur the measurement of modified nucleosides. Artifacts might occur with the use of any of the measurement techniques. The use of proper experimental conditions might avoid artifactual formation of products in DNA. This article reviews mechanistic aspects of oxidative damage to DNA and recent developments in the measurement of this type of damage using chromatographic and mass spectrometric techniques. Published by Elsevier Science, Inc.Öğe Genomic DNA of Nostoc commune (Cyanobacteria) becomes covalently modified during long-term (decades) desiccation but is protected from oxidative damage and degradation(Oxford Univ Press, 2003) Shirkey, B; McMaster, NJ; Smith, SC; Wright, DJ; Rodriguez, H; Jaruga, P; Birincioglu, MGenomic DNA of Nostoc commune ( Cyanobacteria) became covalently modified during decades of desiccation. Amplification of gene loci from desiccated cells required pretreatment of DNA with N-phenacylthiazolium bromide, a reagent that cleaves DNA- and protein-linked advanced glycosylation end-products. DNA from 13 year desiccated cells did not show any higher levels of the commonly studied oxidatively modified DNA damage biomarkers 8-hydroxyguanine, 8-hydroxyadenine and 5-hydroxyuracil, compared to commercially available calf thymus DNA. Different patterns of amplification products were obtained with DNA from desiccated/ rehydrating cells and a liquid culture derived from the dried material, using the same set of primers. In contrast, a reproducible fingerprint was obtained, irrespective of time of rehydration of the DNA, using a primer (5'-GWCWATCGCC-3') based upon a highly iterated palindromic repeat sequence present in the genome. In vitro, the desiccation of cccDNA led to loss of supercoiling, aggregation, loss of resolution during agarose gel electrophoresis and loss of transformation and transfection efficiency. These changes were minimized when DNA was desiccated and stored in the presence of trehalose, a non-reducing disaccharide present in Nostoc colonies. The response of the N. commune genome to desiccation is different from the response of the genomes of cyanobacteria and Deinococcus radiodurans to ionizing radiation.Öğe Mass spectrometric assays for the tandem lesion 8,5?-cyclo-2?-deoxyguanosine in mammalian DNA(Amer Chemical Soc, 2002) Jaruga, P; Birincioglu, M; Rodriguez, H; Dizdaroglu, M8,5'-Cyclopurine 2'-deoxynucleosides are among the major lesions in DNA that are formed by attack of hydroxyl radical. These compounds represent a concomitant damage to both sugar and base moieties of the same nucleoside and thus can be considered tandem lesions. Because of the presence of a covalent bond between the sugar and purine moieties, these tandem lesions are not repaired by base excision repair but by nucleotide excision repair. Thus, they may play a role in diseases with defective nucleotide excision repair. We recently reported the identification and quantification of 8,5'-cyclo-2'-deoxyadenosine (8,5'-cdAdo) in DNA by liquid chromatography/mass spectrometry with the isotope dilution technique (LC/IDMS) [Dizdaroglu, M., Jaruga, P., and Rodriguez, H. (2001) Free Radical Biol. Med. 30, 774-784]. In the present work, we investigated the measurement of 8,5'-cyclo-2'-deoxyguanosine (8,5'-cdGuo) in DNA by LC/IDMS. A methodology was developed for the separation of both (5'R)- and (5'S)-diastereomers of this compound in enzymic hydrolysates of DNA. The mass spectra were recorded using an atmospheric pressure ionization-electrospray process in the positive ionization mode. For quantification, stable isotope-labeled analogues of (5'R)-8,5'-cdGuo and (5'S)-8,5'-cdGuo were prepared and isolated by semipreparative LC to be used as internal standards. The sensitivity level of LC/MS in the selected ion monitoring mode (LC/MS-SIM) was determined to be approximately 15 fmol of these compounds on the LC column. The yield of 8,5'-cdGuo was measured in DNA exposed in aqueous solution to ionizing radiation at doses from 2.5 to 40 Gy. For comparison, gas chromatography/mass spectrometry with the isotope dilution technique (GC/IDMS) was also employed to measure both (5'R)-8,5'-cdGuo and (5'S)-8,5'-cdGuo in DNA. Both techniques yielded nearly identical results. The radiation chemical yield of 8,5'-cdGuo was similar to those of other major purine-derived lesions in DNA. The sensitivity level of GC/MS-SIM was determined to be significantly greater than that of LC/MS-SIM (1 vs 15 fmol). The background levels of (5'R)-8,5'-cdGuo and (5'S)-8,5'-cdGuo were measured in calf thymus DNA and in DNA samples isolated from three different types of cultured human cells. The levels of (5'R)-8,5'-cdGuo and (5'S)-8,5'-cdGuo were approximately 2 lesions/10(6) DNA nucleosides and 10 lesions/10(6) DNA nucleosides, respectively. No significant differences between tissues were observed in terms of these background levels. The results showed that both LC/IDMS and GC/IDMS are well suited for the sensitive detection and precise quantification of both (5'R)-8,5'-cdGuo and (5'S)-8,5'-cdGuo in DNA.Öğe Measurement of oxidative DNA damage by liquid chromatography/mass spectrometry(Pergamon-Elsevier Science Ltd, 2002) Jaruga, P; Birincioglu, M; Rodriguez, H; Dizdaroglu, M[Abstract Not Available]
