8-hydroxyadenine and 8-hydroxyguanine

To understand the structural basis of the recognition and removal of specific mismatched bases in double-stranded DNAs by the DNA repair glycosylase MutY, a series of structural and functional analyses have been conducted. MutY is a 39-kDa enzyme from Escherichia coli, which to date has been refractory to structural determination in its native, intact conformation. However, following limited proteolytic digestion, it was revealed that the MutY protein is composed of two modules, a 26-kDa domain that retains essential catalytic function (designated p26MutY) and a 13-kDa domain that is implicated in substrate specificity and catalytic efficiency. Several structures of the 26-kDa domain have been solved by X-ray crystallographic methods to a resolution of up to 1.2 A. The structure of a catalytically incompetent mutant of p26MutY complexed with an adenine in the substrate-binding pocket allowed us to propose a catalytic mechanism for MutY. Since reporting the structure of p26MutY, significant progress has been made in solving the solution structure of the noncatalytic C-terminal 13-kDa domain of MutY by NMR spectroscopy. The topology and secondary structure of this domain are very similar to that of MutT, a pyrophosphohydrolase. Molecular modeling techniques employed to integrate the two domains of MutY with DNA suggest that MutY can wrap around the DNA and initiate catalysis by potentially flipping adenine and 8-oxoguanine out of the DNA helix.

Topics: Adenine; Amino Acid Sequence; Bacterial Proteins; Base Pair Mismatch; Carbon-Oxygen Lyases; Catalysis; Catalytic Domain; Deoxyribonuclease IV (Phage T4-Induced); DNA Damage; DNA Glycosylases; DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase; DNA, Bacterial; Escherichia coli; Escherichia coli Proteins; Guanine; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; N-Glycosyl Hydrolases; Phosphoric Monoester Hydrolases; Protein Conformation; Protein Structure, Tertiary; Pyrophosphatases; Sequence Alignment; Sequence Homology, Amino Acid; Structure-Activity Relationship; Substrate Specificity

Recently, several papers reported an artifactual formation of a number of modified bases from intact DNA bases during derivatization of DNA hydrolysates to be analyzed by gas chromatography-mass spectrometry (GC/MS). These reports dealt with 8-hydroxyguanine (8-OH-Gua), 5-hydroxycytosine (5-OH-Cyt), 8-hydroxyadenine (8-OH-Ade), 5-hydroxymethyluracil (5-OHMeUra) and 5-formyluracil that represent only a small percentage of the 20 or so modified DNA bases that can be analyzed by GC/MS. Removal of intact DNA bases by prepurification of calf thymus DNA hydrolysates using HPLC was shown to prevent artifactual formation of these modified bases during derivatization. It needs to be emphasized that the procedures for hydrolysis of DNA and derivatization of DNA hydrolysates used in these papers substantially differed from the established procedures previously described. Furthermore, a large number of relevant papers reporting the levels of these modified bases in DNA of various sources have been ignored. Interestingly, the levels of modified bases reported in the literature were not as high as those reported prior to prepurification. Most values for the level of 5-OH-Cyt were even lower than the level measured after prepurification. Levels of 8-OH-Ade were quite close to, or even the same as, or smaller than the level reported after prepurification. The same holds true for 5-OHMeUra and 8-OH-Gua. All these facts raise the question of the validity of the claims about the measurement of these modified DNA bases by GC/MS. A recent paper reported a complete destruction of 2, 6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy-Gua) and 4,6-diamino-5-formamidopyrimidine (FapyAde) by formic acid under the conditions of DNA hydrolysis prior to GC/MS. The complete destruction of FapyGua and FapyAde by formic acid is in disagreement with the data on these compounds in the literature. These two compounds were measured by GC/MS following formic acid hydrolysis for many years in our laboratory and by other researchers with no difficulties. These facts clearly raise the question of the validity of the claims made about the previous measurements of these compounds by GC/MS.

Topics: Adenine; Animals; Artifacts; Base Pairing; Cattle; Cytosine; DNA; DNA Damage; DNA Repair; Gas Chromatography-Mass Spectrometry; Guanine; Humans; Oxidation-Reduction; Oxidative Stress; Pentoxyl; Pyrimidines; Temperature; Thymus Gland

In the present study, we have studied the level of oxidative DNA base damage in lymphocytes of HIV-infected intravenous drug users (IDUs) and a seronegative control group. Chromatin was isolated from the lymphocytes and then analyzed by gas chromatography/isotope-dilution mass spectrometry with selected-ion monitoring (GC/IDMS-SIM). Significantly greater levels of four oxidatively modified DNA bases were observed in chromatin samples from the symptomatic HIV-infected patients than in those from the seronegative patients. These were 5-hydroxyuracil, 5-hydroxycytosine, 8-hydroxyadenine and 8-hydroxyguanine. In the case of 5-hydroxyuracil and 8-hydroxyguanine, a statistically significant difference was also found between the control group and the asymptomatic HIV-positive patients. These results suggest that oxidative stress may play an important role in the pathogenesis of acquired immune deficiency syndrome (AIDS), and that administration of antioxidant drugs to HIV-infected patients may offer protection against AIDS-related carcinogenesis.

Topics: Adenine; Adolescent; Adult; Chromatin; Cytosine; DNA Damage; Guanine; HIV Infections; Humans; Lymphocytes; Male; Oxidation-Reduction; Oxidative Stress; Substance Abuse, Intravenous; Uracil

T4 RNA ligase 2 (Rnl2) repairs 3'-OH/5'-PO4 nicks in duplex nucleic acids in which the broken 3'-OH strand is RNA. Ligation entails three chemical steps: reaction of Rnl2 with ATP to form a covalent Rnl2-(lysyl-Nζ)-AMP intermediate (step 1); transfer of AMP to the 5'-PO4 of the nick to form an activated AppN- intermediate (step 2); and attack by the nick 3'-OH on the AppN- strand to form a 3'-5' phosphodiester (step 3). Here we used rapid mix-quench methods to analyze the kinetic mechanism and fidelity of single-turnover nick sealing by Rnl2-AMP. For substrates with correctly base-paired 3'-OH nick termini, kstep2 was fast (9.5 to 17.9 sec(-1)) and similar in magnitude to kstep3 (7.9 to 32 sec(-1)). Rnl2 fidelity was enforced mainly at the level of step 2 catalysis, whereby 3'-OH base mispairs and oxoguanine, oxoadenine, or abasic lesions opposite the nick 3'-OH elicited severe decrements in the rate of 5'-adenylylation and relatively modest slowing of the rate of phosphodiester synthesis. The exception was the noncanonical A:oxoG base pair, which Rnl2 accepted as a correctly paired end for rapid sealing. These results underscore (1) how Rnl2 requires proper positioning of the 3'-terminal ribonucleoside at the nick for optimal 5'-adenylylation and (2) the potential for nick-sealing ligases to embed mutations during the repair of oxidative damage.

Topics: Adenine; Apurinic Acid; Base Pairing; Base Sequence; Escherichia coli; Guanine; Kinetics; RNA Ligase (ATP); RNA, Double-Stranded; Viral Proteins

While research supports amyloid-β (Aβ) as the etiologic agent of Alzheimer's disease (AD), the mechanism of action remains unclear. Evidence indicates that adducts of RNA caused by oxidation also represent an early phenomenon in AD. It is currently unknown what type of influence these two observations have on each other, if any. We quantified five RNA adducts by gas chromatography/mass spectroscopy across five brain regions from AD cases and age-matched controls. We then used a reductive directed analysis to compare the RNA adducts to common indices of AD neuropathology and various pools of Aβ. Using data from four disease-affected brain regions (Brodmann's Area 9, hippocampus, inferior parietal lobule, and the superior and middle temporal gyri), we found that the RNA adduct 8-hydroxyguanine (8-OHG) decreased, while 8-hydroxyadenine (8-OHA) increased in AD. The cerebellum, which is generally spared in AD, did not show disease related changes, and no RNA adducts correlated with the number of plaques or tangles. Multiple regression analysis revealed that SDS-soluble Aβ(42) was the best predictor of changes in 8-OHG, while formic acid-soluble Aβ(42) was the best predictor of changes in 8-OHA. This study indicates that although there is a connection between AD related neuropathology and RNA oxidation, this relationship is not straightforward.

Topics: Adenine; Aged, 80 and over; Alzheimer Disease; Amyloid beta-Peptides; Brain; Case-Control Studies; DNA Damage; Enzyme-Linked Immunosorbent Assay; Female; Gas Chromatography-Mass Spectrometry; Guanine; Humans; Male; Neurofibrillary Tangles; Oxidation-Reduction; Plaque, Amyloid; RNA

Topics: Adenine; Animals; Base Pair Mismatch; DNA Mismatch Repair; DNA-Directed DNA Polymerase; Guanine; Humans

The blockage of transcription elongation by RNA polymerase II (RNAPII) at DNA lesions on the transcribed strand is a serious challenge to accurate transcription. Transcription-coupled DNA repair (TCR), which is assumed to be initiated by the blockage of transcription, rapidly removes lesions on the transcribed strand of expressed genes and allows the resumption of transcription. Although helix-distorting bulky damage such as a cyclobutane pyrimidine dimer is known to block transcription elongation and to be repaired by TCR, it is not clear whether oxidative DNA lesions are repaired by TCR. First, we examined whether transcription elongation by RNAPII is stalled at sites of 2-hydroxyadenine (2-OH-A), 8-oxoadenine (8-oxoA), 8-oxoguanine (8-oxoG), or thymine glycol (Tg) on the transcribed strand. Our results indicate that RNAPII incorporated nucleotides opposite the lesions and then stalled. In addition, we found that transcription elongation factor TFIIS (SII) enabled RNAPII to bypass 8-oxoG but not the other types of damage, while transcription initiation and elongation factor TFIIF did not bypass 8-oxoG. These results suggest that SII is important for preventing cellular death due to oxidative DNA damage, assisting RNAPII to bypass 8-oxoG.

Topics: Adenine; Base Sequence; DNA Damage; DNA Repair; Guanine; HeLa Cells; Humans; Models, Chemical; Models, Genetic; Molecular Sequence Data; Oxygen; RNA Polymerase II; Thymine; Transcription, Genetic; Transcriptional Elongation Factors

Formamidopyrimidine DNA glycosylase (Fpg) is a DNA glycosylase with an associated AP lyase activity. As a DNA repair enzyme, Fpg excises several modified bases from DNA associated with exposure to oxidizing agents such as free radicals. Experiments in many laboratories have been limited by the availability of the enzyme, and its production required at least a week of work to complete its purification. We have devised a new method that decreases the time and expense of purification of Fpg that should render this protein accessible to any laboratory. Fpg was subcloned into a gamma P(L) promoter-containing vector (pRE) and overproduced in the appropriate Escherichia coli host cells to about 25% of the total cellular protein. Fpg was purified to homogeneity in a simple two-step procedure with a 50% saving in time when compared to the previously known procedure. Comparative studies showed that the excision of 8-hydroxyguanine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 4,6-diamino-5-formamidopyrimidine, and to a lesser extent, 8-hydroxyadenine was virtually identical for the Fpg purified using this method and for the Fpg purified by the original method. Therefore, this method should prove useful for a large number of laboratories and further research on oxidative DNA damage.

Topics: Adenine; Carboxymethylcellulose Sodium; Chromatography, DEAE-Cellulose; Cloning, Molecular; DNA Damage; DNA Repair; DNA-Formamidopyrimidine Glycosylase; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Gas Chromatography-Mass Spectrometry; Gene Expression Regulation, Bacterial; Genetic Vectors; Guanine; Kinetics; Nitrous Oxide; Pyrimidines; Recombinant Proteins; Substrate Specificity; Temperature

Mutagenic oxidative DNA base damage increases with age in prostatic tissue. Various factors may influence this increase including: increased production of reactive oxygen species, increased susceptibility to oxidative stress, alterations in detoxifying enzyme levels or defects in DNA repair. Using liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry, we show increased levels of oxidative DNA base lesions, 8-hydroxyguanine (8-oxoG), 8-hydroxyadenine (8-oxoA) and 5-hydroxycytosine (5OHC) over the baseline in PC-3 and DU-145 prostate cancer cells following exposure to ionizing radiation and a repair period. Nuclear extracts from PC-3 and DU-145 prostate cancer cell lines are defective in the incision of 8-oxoG, 5OHC and thymine glycol (TG) relative to the non-malignant prostate cell line. Consistent with reduced expression of OGG1 2a, incision of 8-oxoG is reduced in PC-3 and DU-145 mitochondrial extracts. We also show a correlation between severely defective incision of TG and 5OHC and reduced levels of NTH1 in PC-3 mitochondria. The antioxidant enzymes, glutathione peroxidase (GPx), catalase and superoxide dismutases (SOD1, SOD2), have altered expression patterns in these cancer cell lines. Genetic analysis of the OGG1 gene reveals that both PC-3 and DU-145 cell lines harbor polymorphisms associated with a higher susceptibility to certain cancers. These data suggest that the malignant phenotype in PC-3 and DU-145 cell lines may be associated with defects in base excision repair and alterations in expression of antioxidant enzymes.

Topics: Adenine; Antioxidants; Blotting, Western; Catalase; Cell Division; Cell Line, Tumor; Cell Nucleus; Comet Assay; Cytosine; DNA; DNA Damage; Dose-Response Relationship, Drug; Electron Transport Complex IV; Gas Chromatography-Mass Spectrometry; Glutathione Peroxidase; Guanine; Humans; Kinetics; Male; Mitochondria; Oxygen; Polymorphism, Genetic; Prostatic Neoplasms; Superoxide Dismutase; Time Factors

Cockayne syndrome (CS) is a genetic human disease with clinical symptoms that include neurodegeneration and premature aging. The disease is caused by the disruption of CSA, CSB, or some types of xeroderma pigmentosum genes. It is known that the CSB protein coded by the CS group B gene plays a role in the repair of 8-hydroxyguanine (8-OH-Gua) in transcription-coupled and non-strand discriminating modes. Recently we reported a defect of CSB mutant cells in the repair of another oxidatively modified lesion 8-hydroxyadenine (8-OH-Ade). We show here that primary fibroblasts from CS patients lack the ability to efficiently repair these particular types of oxidatively induced DNA damages. Primary fibroblasts of 11 CS patients and 6 control individuals were exposed to 2 Gy of ionizing radiation to induce oxidative DNA damage and allowed to repair the damage. DNA from cells was analyzed using liquid chromatography/isotope dilution mass spectrometry to measure the biologically important lesions 8-OH-Gua and 8-OH-Ade. After irradiation, no significant change in background levels of 8-OH-Gua and 8-OH-Ade was observed in control human cells, indicating their complete cellular repair. In contrast, cells from CS patients accumulated significant amounts of these lesions, providing evidence for a lack of DNA repair. This was supported by the observation that incision of 8-OH-Gua- or 8-OH-Ade-containing oligodeoxynucleotides by whole cell extracts of fibroblasts from CS patients was deficient compared to control individuals. This study suggests that the cells from CS patients accumulate oxidatively induced specific DNA base lesions, especially after oxidative stress. A deficiency in cellular repair of oxidative DNA damage might contribute to developmental defects in CS patients.

Topics: Adenine; Adult; Cell Line; Child; Child, Preschool; Cockayne Syndrome; DNA; DNA Damage; DNA Repair; Endonucleases; Exonucleases; Female; Guanine; Humans; Infant; Male; Oxidative Stress

Replication of DNA containing 8-oxo-7,8-dihydroguanine (8oxoG) can generate 8oxoG/A base pairs which, if uncorrected, lead to G-->T transversions. It is generally accepted that the repair of these promutagenic base pairs in human cells is initiated by the MutY DNA glycosylase homolog (hMYH). Here we provide biochemical evidence that human cell extracts perform base excision repair (BER) on both DNA strands of an 8oxoG/A mismatch. At early repair times the specificity of nucleotide incorporation indicates a preferential insertion of C opposite 8oxoG leading to the formation of 8oxoG/C pairs. This is followed by repair synthesis on the opposite DNA strand that is consistent with hOGG1-mediated correction of 8oxoG/C to G/C. Repair synthesis on either strand is completely inhibited by aphidicolin suggesting that a replicative DNA polymerase is involved in the gap filling. This is the first demonstration that repair of 8oxoG/A base pairs is by two BER events likely mediated by Poldelta/epsilon. We suggest that the Poldelta/epsilon-mediated BER is the general mode of repair when BER lesions are formed at replication forks.

Topics: Adenine; Aphidicolin; Base Pair Mismatch; Base Pairing; Cell Extracts; DNA; DNA Primers; DNA Repair; Drug Resistance; Enzyme Inhibitors; Guanine; HeLa Cells; Humans; Nucleic Acid Synthesis Inhibitors

Many cellular functions including gene expression and chromosome structure are highly dependent upon the precise recognition and binding of specific DNA elements by regulatory and structural proteins. DNA damage that alters protein/DNA interactions therefore has the potential to disrupt normal cellular functions including proliferation. As a model to examine the interaction of proteins with damaged DNA, the binding of AP-1 transcription factor to cognate DNA elements with 8-oxoadenine, 8-oxoguanine and abasic sites was studied by gel mobility shift analysis. Of the three types of DNA damage only 8-oxoadenine was without effect on AP-1 binding. A single 8-oxoguanine could partially inhibit AP-1 binding when located at specific positions within and even adjacent to the conserved AP-1 binding sequence. Abasic site damage also demonstrated a position effect but with more overall inhibition. When 8-oxoguanine and abasic sites were combined to model the multiple damage sites produced by ionizing radiation there was a cumulative loss of AP-1 binding that appeared to be synergistic. These results suggest protein/DNA interactions can be quite sensitive to the site, degree, and type of DNA damage, even relatively minor modifications.

Topics: Adenine; Binding Sites; DNA; DNA Damage; DNA-Binding Proteins; Guanine; Promoter Regions, Genetic; Protein Binding; Transcription Factor AP-1

Cockayne syndrome (CS) is a human disease characterized by sensitivity to sunlight, severe neurological abnormalities, and accelerated aging. CS has two complementation groups, CS-A and CS-B. The CSB gene encodes the CSB protein with 1493 amino acids. We previously reported that the CSB protein is involved in cellular repair of 8-hydroxyguanine, an abundant lesion in oxidatively damaged DNA and that the putative helicase motif V/VI of the CSB may play a role in this process. The present study investigated the role of the CSB protein in cellular repair of 8-hydroxyadenine (8-OH-Ade), another abundant lesion in oxidatively damaged DNA. Extracts of CS-B-null cells and mutant cells with site-directed mutation in the motif VI of the putative helicase domain incised 8-hydroxyadenine in vitro less efficiently than wild type cells. Furthermore, CS-B-null and motif VI mutant cells accumulated more 8-hydroxyadenine in their genomic DNA than wild type cells after exposure to gamma-radiation at doses of 2 or 5 Gy. These results suggest that the CSB protein contributes to cellular repair of 8-OH-Ade and that the motif VI of the putative helicase domain of CSB is required for this activity.

Topics: Adenine; DNA Helicases; DNA Repair; DNA Repair Enzymes; Gamma Rays; Guanine; Humans; Poly-ADP-Ribose Binding Proteins

Increased intestinal bile acids as a possible consequence of a high fat/meat, low fiber diet are believed to play an important role in the formation of colon cancer. Interactions of bile salts particularly secondary bile acids with different cell components including DNA may contribute to carcinogenesis. To further investigate DNA damage by bile salts, we assessed the effects of a bile salt mixture containing deoxycholate and chenodeoxycholate on base hydroxylation in Chelex-treated DNA from calf thymus as a model of human colonic mucosal DNA in the presence and absence of reactive oxygen metabolites (ROM).. Chelex-treated DNA from calf thymus (to remove residual iron impurities) was incubated with different bile salt concentrations (4 microM, 4.0 mM) (20.0% deoxycholate, 21.0% chenodeoxycholate) in the presence and absence of an OH generating system (25 microM FeCl3, 50 microM H2O2, 100 microM nitrilotriacetic acid) for 18 h (37 degrees C). After hydrolyzation, lyophilization and derivatization hydroxylated DNA bases were characterised and quantitated with gas chromatography-mass spectrometry (GS-MS) and SIM analysis. Two concentration ranges of bile salts were used, micromolar concentrations being present in plasma, millimolar in the gut lumen.. In the absence of ROM Chelex-treated DNA preparations contain only small amounts of hydroxylated base products. Bile salts at 4.0 mM significantly increased the amounts of 5-OH uracil and cis-thymine glycol. In the presence of ROM bile salts at 4.0 microM increased the production of 8-OH adenine and 8-OH guanine whereas bile salts at 4.0 mM inhibited ROM-induced base hydroxylation.. In the absence of ROM millimolar concentrations of a bile salt mixture with deoxycholate and chenodeoxycholate increase basal (spontaneous) DNA hydroxylation, whereas, they are without effects at micromolar concentrations. In the presence of ROM micromolar concentrations enhance oxidative DNA damage and millimolar concentrations were inhibitory. These results support the view that bile acids may cause oxidative DNA damage depending on their concentrations and the surrounding conditions both directly (enhancement of basal hydroxylation) and indirectly (enhancement of ROM-induced hydroxylation).

Topics: Adenine; Animals; Cattle; Chenodeoxycholic Acid; Colon; Colonic Neoplasms; Deoxycholic Acid; DNA; DNA Damage; Gas Chromatography-Mass Spectrometry; Gastrointestinal Agents; Guanine; Humans; Hydroxylation; Intestinal Mucosa; Models, Chemical; Reactive Oxygen Species; Salicylates; Thymus Gland

Werner syndrome (WS) is a premature aging disorder where the affected individuals appear much older than their chronological age. The single gene that is defective in WS encodes a protein (WRN) that has ATPase, helicase and 3'-->5' exonuclease activities. Our laboratory has recently uncovered a physical and functional interaction between WRN and the Ku heterodimer complex that functions in double-strand break repair and V(D)J recombination. Importantly, Ku specifically stimulates the exonuclease activity of WRN. We now report that Ku enables the Werner exonuclease to digest through regions of DNA containing 8-oxoadenine and 8-oxoguanine modifications, lesions that have previously been shown to block the exonuclease activity of WRN alone. These results indicate that Ku significantly alters the exonuclease function of WRN and suggest that the two proteins function concomitantly in a DNA damage processing pathway. In support of this notion we also observed co-localization of WRN and Ku, particularly after DNA damaging treatments.

Topics: Adenine; Antigens, Nuclear; Cell Line; Cell Nucleus; DNA; DNA Damage; DNA Helicases; DNA-Binding Proteins; Exodeoxyribonucleases; Exonucleases; Guanine; HeLa Cells; Humans; Ku Autoantigen; Nuclear Proteins; RecQ Helicases; Replication Protein A; Werner Syndrome Helicase

We measured concentrations and ratios of mutagenic (8-OH) lesions to putatively nonmutagenic formamidopyrimidine (Fapy) lesions of adenine (Ade) and guanine (Gua) to elucidate radical (.OH)-induced changes in DNA of normal, normal from cancer, and cancer tissues of the prostate. The relationship between the lesions was expressed using the mathematical model log(10)[(8-OH-Ade + 8-OH-Gua)/(FapyAde + FapyGua)]. Logistic regression analysis of the log ratios for DNA of normal and cancer tissues discriminated between the two tissue groups with high sensitivity and specificity. Correlation analysis of log ratios for normal prostates revealed a highly significant increase in the proportion of mutagenic base lesions with age. Data from correlation analysis of the log ratios for normal tissues from cancer were consistent with an age-dependent, dose-response relationship. The slopes for both correlations intersected at approximately 61 years, an age when prostate cancer incidence is known to rise sharply. The age-related increase in the proportion of.OH-induced mutagenic base lesions is likely a significant factor in prostate cancer development.

Topics: Adenine; Age Factors; Cell Transformation, Neoplastic; DNA; DNA Damage; DNA, Neoplasm; Gas Chromatography-Mass Spectrometry; Guanine; Humans; Hydroxyl Radical; Logistic Models; Male; Middle Aged; Models, Biological; Prostate; Prostatic Neoplasms; Pyrimidines

Type II topoisomerases are essential enzymes that are also the primary cellular targets for a number of important anticancer drugs. These drugs act by increasing levels of topoisomerase II-mediated DNA cleavage. Recent studies indicate that endogenous forms of DNA damage, such as abasic sites and base mismatches, also stimulate the DNA scission activity of the enzyme. To extend our understanding of how type II topoisomerases react to DNA damage, the effects of abasic sites, and oxidized and alkylated bases on DNA cleavage mediated by human topo-isomerase IIalpha and beta were determined. Based on experiments that incorporated random abasic sites into plasmid DNA, human type II enzymes can locate lesions even within a background of several thousand undamaged base pairs. As determined by experiments that utilized site-specific forms of DNA lesions, oxidized or monoalkylated purines that allow base pairing and induce little distortion in the double helix have modest effects on topoisomerase II-mediated DNA cleavage. In contrast, 1,N(6)-ethenoadenine, a bulky lesion that disrupts base pairing, enhanced DNA cleavage approximately 10-fold. 1,N(6)-Ethenoadenine is the first lesion found to rival the stimulatory effects of apurinic sites on the DNA scission activity of eukaryotic type II topoisomerases.

Topics: Adenine; Alkylation; Apurinic Acid; Base Sequence; DNA; DNA Adducts; DNA Damage; DNA Repair; DNA Topoisomerases, Type II; DNA-Binding Proteins; Guanine; Humans; Isoenzymes; Mutation; Oligonucleotides; Oxidation-Reduction; Plasmids; Sensitivity and Specificity

Individuals with mutations in the WRN gene suffer from Werner syndrome, a disease with early onset of many characteristics of normal aging. The WRN protein (WRNp) functions in DNA metabolism, as the purified polypeptide has both 3'-->5' helicase and 3'-->5' exonuclease activities. In this study, we have further characterized WRNp exonuclease activity by examining its ability to degrade double-stranded DNA substrates containing abnormal and damaged nucleo-tides. In addition, we directly compared the 3'-->5' WRNp exonuclease activity with that of exo-nuclease III and the Klenow fragment of DNA polymerase I. Our results indicate that the presence of certain abnormal bases (such as uracil and hypoxanthine) does not inhibit the exonuclease activity of WRNp, exo-nuclease III or Klenow, whereas other DNA modifications, including apurinic sites, 8-oxoguanine, 8-oxoadenine and cholesterol adducts, inhibit or block WRNp. The ability of damaged nucleo-tides to inhibit exonucleolytic digestion differs significantly between WRNp, exonuclease III and Klenow, indicating that each exonuclease has a distinct mechanism of action. In addition, normal and modified DNA substrates are degraded similarly by full-length WRNp and an N-terminal fragment of WRNp, indicating that the specificity for this activity lies mostly within this region. The biochemical and physiological significance of these results is discussed.

Topics: Adenine; Base Sequence; DNA; DNA Adducts; DNA Helicases; DNA Polymerase I; Exodeoxyribonucleases; Exonucleases; Guanine; Hypoxanthine; Mutation; Oxidation-Reduction; RecQ Helicases; Substrate Specificity; Uracil; Werner Syndrome Helicase

A general procedure to obtain tetra-substituted uric acid by stepwise N-alkylation is described. 2,6-Dichloropurine (1) was condensed with 1-propanol by Mitsunobu reaction to give 9-propyl congener (2). Treatment of 2 with ammonia gave adenine derivative (4a), which was converted to the 8-oxoadenine (5b) in 3 steps. Methylation of 5b proceeded site-specifically to give 6-amino-2-chloro-7,8-dihydro-7-methyl-9-propylpurin-8-one (6) as a sole product. Compound 6 was successively treated with NaNO2 and iodomethane to give 2-chloro-1,6,7,8-tetrahydro-1,7-dimethyl-9-propylpurin-6,8-dione (9) accompanied by the O6-methyl product (8) in 75% and 6.9%, respectively. After nucleophilic substitution of 9 with NaOAc, the product (11) was reacted with iodomethane to give the uric acid (12) and the 2-methoxy product (13) in 46% and 15.5%, respectively. However, the reaction of 11 with the benzylating agents gave only O-benzyl products (14a,b).

Topics: 1-Propanol; Adenine; Alkylation; Ammonia; Guanine; Hydrocarbons, Iodinated; Mass Spectrometry; Methylation; Purines; Uric Acid

Structural changes in a 25-base DNA strand, induced by single 8-oxo-guanine or 8-oxo-adenine substitutions, were shown by using Fourier transform-infrared spectroscopy with multivariate statistics. Pronounced differences were demonstrated between the parent and derivatives with respect to base interactions and changes in the phospho-deoxyribose backbone. The greatest degree of change in the backbone likely occurred immediately adjacent to the 8-oxo group, potentially altering the stereochemistry at a distance. The 8-oxo lesions, formed from reactive oxygen species (e.g., hydroxyl radicals), may appreciably alter the conformational properties of strands at the replication fork, thus affecting the selectivity of polymerases, the proofreading capability of repair enzymes, and the fidelity of the transcriptional machinery.

Topics: Adenine; DNA; DNA Damage; Guanine; Hydroxyl Radical; Multivariate Analysis; Nucleic Acid Conformation; Oligodeoxyribonucleotides; Spectroscopy, Fourier Transform Infrared

Recently, an artifactual formation of a number of modified DNA bases has been alleged during derivatization of DNA hydrolysates to be analyzed by gas chromatography-mass spectrometry (GC-MS). These modified bases were 8-hydroxyguanine (8-OH-Gua), 5-hydroxycytosine (5-OH-Cyt), 8-hydroxyadenine (8-OH-Ade), 5-hydroxymethyluracil (5-OHMeUra), and 5-formyluracil, which represent only a small percentage of more than 20 modified DNA bases that can be analyzed by GC-MS. However, relevant papers reporting the levels of these modified bases in DNA of various sources have not been cited, and differences in experimental procedures have not been discussed. We investigated the levels of modified bases in calf thymus DNA by GC-MS using derivatization at three different temperatures. The results obtained with GC/isotope-dilution MS showed that the levels of 5-OH-Cyt, 8-OH-Ade, 5-OH-Ura, and 5-OHMeUra were not affected by increasing the derivatization temperature from 23 degrees C to 120 degrees C. The level of 8-OH-Gua was found to be higher at 120 degrees C. However, this level was much lower than those reported previously. Formamidopyrimidines were readily analyzed in contrast to some recent claims. The addition of trifluoroacetic acid (TFA) adversely affected the levels of pyrimidine-derived lesions, suggesting that TFA is not suitable for simultaneous measurement of both pyrimidine- and purine-derived lesions. The data obtained were also compared with those previously published. Our data and this comparison indicate that no artifactual formation of 5-OH-Cyt, 8-OH-Ade, and 5-OHMeUra occurred under our experimental conditions in contrast to recent claims, and no prepurification of DNA hydrolysates by a tedious procedure is necessary for accurate quantification of these compounds. The artifactual formation of 8-OH-Gua can be eliminated by derivatization at room temperature for at least 2 h, without the use of TFA. The results in this article and their comparison with published data indicate that different results may be obtained in different laboratories using different experimental conditions. The data obtained in various laboratories should be compared by discussing all relevant published data and scientific facts, including differences between experimental conditions used in different laboratories.

Topics: Adenine; Animals; Artifacts; Cattle; Cytosine; DNA; Gas Chromatography-Mass Spectrometry; Guanine; Hydrolysis; Nucleotides; Oxidation-Reduction; Pentoxyl; Trifluoroacetic Acid; Uracil

Recently we found that KMnO4 oxidation of DNA oligomers containing a 7,8-dihydro-8-oxoguanine (8-oxo-G) residue induces damage to the neighboring base residues; other modified bases, 7,8-dihydro-8-oxoadenine (8-oxo-A) and 5-hydroxyuracil (5-oh-U), show similar behavior in DNA. The present study indicated that the ability to induce damage, which could also occur by the oxidation of a 5-oh-C residue, was low as in the case of 5-oh-U. On the other hand, in order to examine the pathways and the intermediates for the oxidative degradation of 8-oxo-A, we have carried out the KMnO4 oxidation using an 8-oxo-2'-deoxyadenosine derivative as a model and have determined the structures of the three major products.

Topics: Adenine; Base Sequence; DNA; DNA Damage; Guanine; Oligodeoxyribonucleotides; Oxidation-Reduction; Potassium Permanganate; Uracil

Analysis of oxidative damage to DNA bases by GC-MS enables identification of a range of base oxidation products, but requires a derivatization procedure. However, derivatization at high temperature in the presence of air can cause 'artifactual' oxidation of some undamaged bases, leading to an overestimation of their oxidation products, including 8-hydroxyguanine. Therefore derivatization conditions that could minimize this problem were investigated. Decreasing derivatization temperature to 23 degrees C lowered levels of 8-hydroxyguanine, 8-hydroxyadenine, 5-hydroxycytosine and 5-(hydroxymethyl)uracil measured by GC-MS in hydrolysed calf thymus DNA. Addition of the reducing agent ethanethiol (5%, v/v) to DNA samples during trimethylsilylation at 90 degrees C also decreased levels of these four oxidized DNA bases as well as 5-hydroxyuracil. Removal of guanine from hydrolysed DNA samples by treatment with guanase, prior to derivatization, resulted in 8-hydroxyguanine levels (54-59 pmol/mg of DNA) that were significantly lower than samples not pretreated with guanase, independent of the derivatization conditions used. Only hydrolysed DNA samples that were derivatized at 23 degrees C in the presence of ethanethiol produced 8-hydroxyguanine levels (56+/-8 pmol/mg of DNA) that were as low as those of guanase-pretreated samples. Levels of other oxidized bases were similar to samples derivatized at 23 degrees C without ethanethiol, except for 5-hydroxycytosine and 5-hydroxyuracil, which were further decreased by ethanethiol. Levels of 8-hydroxyguanine, 8-hydroxyadenine and 5-hydroxycytosine measured in hydrolysed calf thymus DNA by the improved procedures described here were comparable with those reported previously by HPLC with electrochemical detection and by GC-MS with prepurification to remove undamaged base. We conclude that artifactual oxidation of DNA bases during derivatization can be prevented by decreasing the temperature to 23 degrees C, removing air from the derivatization reaction and adding ethanethiol.

Topics: Adenine; Animals; Cattle; Cytosine; DNA; DNA Damage; Gas Chromatography-Mass Spectrometry; Guanine; Guanine Deaminase; Oxidation-Reduction; Pentoxyl; Sulfhydryl Compounds; Temperature

Two gel electrophoretic methods are described for detection of 7, 8-dihydro-8-oxoguanine and 7,8-dihydro-8-oxoadenine based on their further oxidation with one-electron oxidants including IrCl62-and IrBr62-. The products of nucleobase oxidation lead to enhanced piperidine-sensitive cleavage and to highly visible stop points in a primer extension assay. 8-oxoG and 8-oxoA lesions may be distinguished by the latter's inability to be oxidized by IrBr62-compared to IrCl62-Comparison is also made to oxidation by MnO4-.

Topics: Adenine; DNA Damage; DNA Primers; Electrophoresis, Polyacrylamide Gel; Guanine; Iridium; Oxidation-Reduction

DNA base oxidation is considered to be a key event associated with disease initiation and progression in humans. Peroxyl radicals (ROO. ) are important oxidants found in cells whose ability to react with the DNA bases has not been characterized extensively. In this paper, the products resulting from ROO. oxidation of the DNA bases are determined by gas chromatography/MS in comparison with authentic standards. ROO. radicals oxidize adenine and guanine to their 8-hydroxy derivatives, which are considered biomarkers of hydroxyl radical (HO.) oxidations in cells. ROO. radicals also oxidize adenine to its hydroxylamine, a previously unidentified product. ROO. radicals oxidize cytosine and thymine to the monohydroxy and dihydroxy derivatives that are formed by oxidative damage in cells. Identical ROO. oxidation profiles are observed for each base when exposed as deoxyribonucleosides, monohomopolymers and base-paired dihomopolymers. These results have significance for the development, utilization and interpretation of DNA base-derived biomarkers of oxidative damage associated with disease initiation and propagation, and support the idea that the mutagenic potential of N-oxidized bases, when generated in cellular DNA, will require careful evaluation. Adenine hydroxylamine is proposed as a specific molecular probe for the activity of ROO. in cellular systems.

Topics: Adenine; Amidines; Base Pairing; Cytosine; Deoxyribonucleosides; DNA; Free Radicals; Guanine; Mass Spectrometry; Oxidation-Reduction; Purines; Pyrimidines; Reactive Oxygen Species; Thymine

Multiple lines of evidence indicate that oxidative stress is a contributor to neuronal death in Alzheimer's disease (AD). The oxidative damage that occurs to DNA may play a role in both normal aging and neurodegenerative diseases, including AD. This is a study of the oxidative damage that occurs in nuclear DNA in the brains of AD patients and cognitively intact, prospectively evaluated, age-matched control subjects. Nuclear DNA from frontal, temporal, and parietal lobes and cerebellum was isolated from 11 control subjects and 9 AD subjects, and oxidized purine and pyrimidine bases were quantitated using gas chromatography/mass spectrometry. Stable isotope-labeled oxidized base analogues were used as internal standards to measure 5-hydroxyuracil, 5-hydroxycytosine, 8-hydroxyadenine, 4,6-diamino-5-formamidopyrimidine (Fapy-adenine), 8-hydroxyguanine, and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy-guanine). Statistically significant elevations of 5-hydroxycytosine, 5-hydroxyuracil, 8-hydroxyadenine, and 8-hydroxyguanine were found in AD brain compared with control subjects (p < 0.05). There was an increased trend in the levels of Fapy-adenine in the AD brain, and Fapy-guanine showed a trend toward higher levels in control brains compared with AD. A generally higher level of oxidative DNA damage was present in neocortical regions than cerebellum. No significant correlation was observed between the oxidized bases and neurofibrillary tangle and senile plaque counts. Our results demonstrate that nuclear DNA damage by oxygen-derived radicals is increased in AD and support the concept that the brain is under increased oxidative stress in AD.

Topics: Adenine; Aged; Aged, 80 and over; Alzheimer Disease; Brain; Cadaver; Cell Nucleus; Cytosine; DNA; Gas Chromatography-Mass Spectrometry; Guanine; Humans; Oxidation-Reduction; Pyrimidines; Reference Values; Tissue Distribution

GC-MS is a widely used tool to measure oxidative DNA damage because of its ability to identify a wide range of base modification products. However, it has been suggested that the derivatization procedures required to form volatile products prior to GC-MS analysis can sometimes produce artifactual formation of certain base oxidation products, although these studies did not replicate previously-used reaction conditions, e.g. they failed to remove air from the derivatization vials. A systematic examination of this problem revealed that levels of 8-hydroxyguanine, 8-hydroxyadenine, 5-hydroxycytosine and 5-(hydroxymethyluracil) in commercial calf thymus DNA determined by GC-MS are elevated by increasing the temperature at which derivatization is performed in our laboratory. In particular, 8-hydroxyguanine levels after silylation at 140 degrees C were raised 8-fold compared to derivatization at 23 degrees C. Experiments on the derivatization of each undamaged base revealed that the artifactual oxidation of guanine, adenine, cytosine and thymine respectively was responsible. Formation of the above products was potentiated by not purging with nitrogen prior to derivatization. Increasing the temperature to 140 degrees C or allowing air to be present during derivatization did not significantly increase levels of the other oxidized bases measured. This work suggests that artifactual oxidation during derivatization is restricted to certain products (8-hydroxyguanine, 8-hydroxyadenine, 5-hydroxycytosine and 5-[hydroxymethyluracil]) and can be decreased by reducing the temperature of the derivatization reaction to 23 degrees C and excluding as much air possible. Despite some recent reports, we were easily able to detect formamidopyrimidines in acid-hydrolyzed DNA. Artifacts of derivatization are less marked than has been claimed in some papers and may vary between laboratories, depending on the experimental procedures used, in particular the efficiency of exclusion of O2 during the derivatization process.

Topics: Adenine; Animals; Artifacts; Cytosine; DNA; DNA Damage; Gas Chromatography-Mass Spectrometry; Guanine; Hydantoins; Hydrolysis; Nitrogen; Oxidation-Reduction; Pentoxyl; Purines; Pyrimidines; Temperature; Time Factors

This aim of this study was to measure the typical free radical-induced products of DNA bases in cellular DNA of cervical cancer tissues directly irradiated by applying brachytherapy to the patients. Significant increases in the amounts of modified bases over the control level were observed in the samples isolated after irradiation for all patients. These increases differed among patients and among products. The repair capacity and/or the amount of hypoxic cells inside the tumor may account for the different levels of modified bases. It is possible that the observed variabilities may account for the differences in clinical responses to brachytherapy.

Topics: Adenine; Brachytherapy; Cytosine; DNA Damage; DNA, Neoplasm; Female; Gas Chromatography-Mass Spectrometry; Guanine; Humans; Oxidation-Reduction; Pyrimidines; Uracil; Uterine Cervical Neoplasms

Hydroxyl radical-induced mutagenic base modifications have been linked to neoplasia in a number of biological systems, including English sole from chemically contaminated urban environments. However, virtually no information exists on the relationship between the mutagenic base modifications and preneoplastic and other lesions found in tumor-free tissues prone to cancer. We studied six hepatic lesions in immature, neoplasm-free English sole exposed to an urban and reference environment and established correlations between the lesion incidence and concentrations of the mutagenic base modifications 8-hydroxyguanine and 8-hydroxyadenine. The lesions were putatively preneoplastic basophilic foci, hepatocellular karyomegaly, megalocytic hepatosis, hepatocellular vacuolar change, hyalin droplet formation, and apoptosis. With the exception of hepatocellular vacuolar change, significant positive correlations were found between the lesions and the mutagenic base modifications. The hydroxyl radical may be a common etiological factor in the formation of the base modifications and hepatic lesions.

Topics: Adenine; Age Factors; Animals; Confounding Factors, Epidemiologic; Fish Diseases; Guanine; Liver Diseases; Liver Neoplasms; Mutagenesis; Precancerous Conditions

Modification of bases in calf thymus DNA by treatment with the antipsoriatic drug anthralin was studied. The products of DNA bases were identified and their yields measured by gas chromatography-mass spectrometry with selected ion monitoring. Treatment of calf thymus DNA with anthralin significantly enhanced the amount of modified bases above control levels. Purine bases were modified to products identical with those known to be typical of DNA damage induced by hydroxyl radicals. The yields of Fapy-adenine, 8-hydroxyadenine, Fapy-guanine, and 8-hydroxyguanine were maximally increased at an anthralin concentration of 75 microM. A variety of structural analogues of anthralin were also tested at 75 microM were either weaker or stronger hydroxylating agents. It is likely that damage to DNA bases induced by anthrones contributes to their antiproliferative activity. The pharmacological implications of these characteristics of the action of anthralin on DNA bases are discussed.

Topics: Adenine; Animals; Anthralin; Cattle; Dermatologic Agents; DNA; DNA Damage; Gas Chromatography-Mass Spectrometry; Guanine; Hydroxylation; Pyrimidines

The extent of DNA modification in cancerous rat live and lung tissues was investigated and compared to their respective normal tissues. Liver tumors were induced by 2-fluorenylacetamide (2-FAA) or N-nitroso-N-2-fluorenylacetamide (N-NO-2-FAA), and lung tumors were induced by sodium nitrite plus trimethylamine. In the DNA samples isolated from these tissues, two pyrimidine-derived and four purine-derived modified DNA bases were identified and quantified by gas chromatography/mass spectrometry with selected-ion monitoring. These compounds were characterized as 5-hydroxyuracil (5-OHUra), thymine glycol (TG), 4,6-diamino-5-formamidopyrimidine (FapyAde), 2,6-diamino-4-hydroxy-5- formamidopyrimidine (FapyGua), 8-hydroxyadenine (8-OHAde), and 8-hydroxyguanine (8-OHGua). Elevated amounts of modified DNA bases were found in most cancerous tissues when compared to the controls. Chemicals used for tumor induction were responsible for inducing DNA lesions that could be promutagenic in vivo and could lead to various types of mutations. When endogenous oxidative damage to DNA during aging was examined, a roughly 2-fold increase of thymine glycol, 8-OHAde and 8-OHGua was found in aged (12 months) rat liver tissues compared to young tissues (1 month). The same results were also found in lung tissues, except that the amount of thymine glycol exhibited more than a 10-fold increase in aged tissues when compared to young tissues. The association of the modified bases with the processes of aging and carcinogenesis deserves further investigation.

Topics: 2-Acetylaminofluorene; Adenine; Aging; Animals; Antineoplastic Agents; Disease Models, Animal; DNA Damage; DNA, Neoplasm; Gas Chromatography-Mass Spectrometry; Guanine; Hydrolysis; Liver; Liver Neoplasms, Experimental; Lung; Lung Neoplasms; Male; Methylamines; Nitrates; Oxidation-Reduction; Pyrimidines; Random Allocation; Rats; Rats, Sprague-Dawley; Rats, Wistar; Thymine; Uracil

A single 7,8-dihydro-8-oxoguanine (G8-OXO; 8-hydroxyguanine) adduct in the lacZ alpha gene of bacteriophage M13 DNA induces a targeted G-->T transversion after replication in Escherichia coli (Biochemistry, 29, 7024-7031 (1990)). This mutation is thought to be due to the facile formation during DNA synthesis of a G8-OXO.base pair, where G8-OXO is in the syn conformation about the deoxyglycosyl bond. A related modified purine, 7,8-dihydro-8-oxoadenine (A8-OXO; 8-hydroxyadenine), is an abundant product found in irradiated and oxidized DNAs. Similar to G8-OXO, as a mononucleoside A8-OXO assumes the syn conformation. This work has assessed the relative mutagenicities of A8-OXO and G8-OXO in the same experimental system. A deoxypentanucleotide containing A8-OXO [d(GCT-A8-OXOG)] was synthesized. After 5'-phosphorylation with [gamma-32P] ATP, the oligomer was ligated into a duplex M13mp19-derived genome at a unique NheI restriction site. Genomes containing either A8-OXO (at position 6275, [+] strand) or G8-OXO (position 6276) were denatured with heat and introduced into E.coli DL7 cells. Analysis of phage DNA from mutant plaques obtained by plating immediately after transformation (infective centers assay) revealed that G8-OXO induced G-->T transversions at an apparent frequency of approximately 0.3%. The frequency and spectrum of mutations observed in DNA sequences derived from 172 mutant plaques arising from the A8-OXO-modified DNA were almost indistiguishable from those generated from transfection of an adenine-containing control genome. We conclude that A8-OXO is at least an order of magnitude less mutagenic than G8-OXO in E.coli cells with normal DNA repair capabilities.

Topics: Adenine; Bacteriophage M13; Base Sequence; Chromatography, High Pressure Liquid; DNA Damage; DNA, Recombinant; Escherichia coli; Guanine; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutagenicity Tests; Mutagens; Oligodeoxyribonucleotides; Oxidation-Reduction; Restriction Mapping; Transformation, Bacterial

DNA of invasive ductal carcinomas from five women was analyzed for structural alterations in the purine nucleotides using gas chromatography-mass spectrometry with selected ion monitoring. The results were compared to those for a normal DNA control. The carcinoma DNA showed dramatically higher concentrations of the base modifications 8-hydroxy-guanine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyadenine. For example, the concentration of total identified base modifications represented a more than 9-fold increase over the control value. Base modifications of this type, which arise from radical-induced hydroxylation and cleavage reactions of the purine ring, likely play a major role in initiation and probably contribute to the further transformation of neoplastic cells in cancer of the female breast.

Topics: Adenine; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cell Transformation, Neoplastic; Chromatography, Gas; DNA; Female; Guanine; Humans; Hydroxylation; Mass Spectrometry; Pyrimidines