An automated Fpg-based FADU method for the detection of oxidative DNA lesions and screening of antioxidants.
ABSTRACT: The oxidation of guanine to 8-oxo-2'-deoxyguanosine (8-oxo-dG) is one of the most abundant and best studied oxidative DNA lesions and is commonly used as a biomarker for oxidative stress. Over the last decades, various methods for the detection of DNA oxidation products have been established and optimized. However, some of them lack sensitivity or are prone to artifact formation, while others are time-consuming, which hampers their application in screening approaches. In this study, we present a formamidopyrimidine glycosylase (Fpg)-based method to detect oxidative lesions in isolated DNA using a modified protocol of the automated version of the fluorimetric detection of alkaline DNA unwinding (FADU) method, initially developed for the measurement of DNA strand breaks (Moreno-Villanueva et al., 2009. BMC Biotechnol. 9, 39). The FADU-Fpg method was validated using a plasmid DNA model, mimicking mitochondrial DNA, and the results were correlated to 8-oxo-dG levels as measured by LC-MS/MS. The FADU-Fpg method can be applied to analyze the potential of compounds to induce DNA strand breaks and oxidative lesions, as exemplified here by treating plasmid DNA with the peroxynitrite-generating molecule Sin-1. Moreover, this method can be used to screen DNA-protective effects of antioxidant substances, as exemplified here for a small-molecule, i.e., uric acid, and a protein, i.e., manganese superoxide dismutase, both of which displayed a dose-dependent protection against the generation of oxidative DNA lesions. In conclusion, the automated FADU-Fpg method offers a rapid and reliable measurement for the detection of peroxynitrite-mediated DNA damage in a cell-free system, rendering it an ideal method for screening the DNA-protective effects of antioxidant compounds.
Project description:Age-related macular degeneration (AMD) is the leading cause of blindness among older adults in the developed world. Oxidative damage to mitochondrial DNA (mtDNA) in the retinal pigment epithelium (RPE) may play a key role in AMD. Measurement of oxidative DNA lesions such as 8-oxo-2'-deoxyguanosine (8-oxo-dG) and 8-oxo-2'-deoxyadenosine (8-oxo-dA) in diseased RPE could provide important insights into the mechanism of AMD development. We have developed a liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry method for simultaneous analysis of 8-oxo-dG and 8-oxo-dA in human retinal DNA. The developed method was applied to the analysis of retinal DNA from 5 donors with AMD and 5 control donors without AMD. In mtDNA, the levels of 8-oxo-dG in controls and AMD donors averaged 170 and 188, and 8-oxo-dA averaged 11 and 17 adducts per 10(6) bases, respectively. In nuclear DNA, the levels of 8-oxo-dG in controls and AMD donors averaged 0.54 and 0.96, and 8-oxo-dA averaged 0.04 and 0.05 adducts per 10(6) bases, respectively. This highly sensitive method allows for the measurement of both adducts in very small amounts of DNA and can be used in future studies investigating the pathophysiological role of 8-oxo-dG and 8-oxo-dA in AMD and other oxidative damage-related diseases in humans.
Project description:To study whether increase in mitochondrial oxidative stress (SOD2 removal) and decrease in mitochondrial DNA repair (Ogg1 dMTS) results into increase in mitochondrial DNA mutation load. Oxidative stress has been suggested to induce mutations in mtDNA. To verify this, we extracted and sequenced (Illumina) mitochondrial DNA from heart Sod2 knockout animals that were also deficient for mitochondrial base-excision repair. The repair deficiency was induced by removing the genomic region encoding for the predicted mitochondrial targeting sequence from endogenous OGG1 (L2 to W23) called Ogg1 dMTS mice, thus excluding the protein from mitochondria. OGG1 is a DNA glycosylase that recognizes and repairs 8-oxo-dG damage from DNA. Oxidative stress can induce 8-oxo-dG lesions, thus we removed the mitochondrial matrix localized superoxide dismutase (SOD2) from these mice to increase the level of oxidative stress. 8-oxo-dG lesion can be mutagenic because some DNA repair polymerases are known to erroneously incorporate adenosine opposite to 8-oxo-dG during replication leading to GC>TA transversion mutations.
Project description:Mitochondrial DNA is replicated and repaired by DNA polymerase gamma (pol gamma), encoded by the POLG gene. The Y955C substitution in POLG leads to autosomal dominant progressive external ophthalmoplegia (PEO) with other severe phenotypes. PEO patients with this mutation can further develop parkinsonism or premature ovarian failure. Mouse and yeast models with this mutation show enhanced amounts of oxidative lesions and increased mtDNA damage. In DNA pol gamma, Tyr955 plays a critical role in catalysis and high fidelity DNA synthesis. 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) is one of the most common oxidative lesions in DNA and can promote transversion mutations. Mitochondria are thought to be a major source of endogenous reactive oxygen species that can react with dG to form 8-oxo-dG as one of the more common products. DNA polymerases can mitigate mutagenesis by 8-oxo-dG through allosteric interactions from amino acid side chains, which limit the anti-conformation of the 8-oxo-dG template base during translesion DNA synthesis. Here, we show that the Y955C pol gamma displays relaxed discrimination when either incorporating 8-oxo-dGTP or translesion synthesis opposite 8-oxo-dG. Molecular modeling and biochemical analysis suggest that this residue, Tyr955, in conjunction with Phe961 helps attenuate the anti-conformation in human pol gamma for error free bypass of 8-oxo-dG and substitution to Cys allows the mutagenic syn conformation. Collectively, these results offer a biochemical link between the observed oxidative stress in model systems and parkinsonism in patients, suggesting that patients harboring the Y955C POLG mutation may undergo enhanced oxidative stress and DNA mutagenesis.
Project description:To study whether increase in mitochondrial oxidative stress (SOD2 removal) and decrease in mitochondrial DNA repair (Ogg1 dMTS) results into increase in mutations in mitochondrial RNA (mtRNA). Oxidative stress has been suggested to induce mutations in mtDNA and as DNA is used as a template in transcription, mutations or 8-oxo-dG on DNA can induce GC>TA transversion accumulation in mtRNA. To verify this, we extracted and sequenced (Illumina) total RNA from heart Sod2 knockout mice alone and mice that were also deficient for mitochondrial base-excision repair. The repair deficiency was induced by removing the genomic region encoding for the predicted mitochondrial targeting sequence from endogenous OGG1 (L2 to W23) called Ogg1 dMTS mice, thus excluding the protein from mitochondria. OGG1 is a DNA glycosylase that recognizes and repairs 8-oxo-dG damage from DNA. Oxidative stress can induce 8-oxo-dG lesions, thus we removed the mitochondrial matrix localized superoxide dismutase (SOD2) from these mice to increase the level of oxidative stress. 8-oxo-dG lesion can be mutagenic because some DNA repair polymerases are known to erroneously incorporate adenosine opposite to 8-oxo-dG during replication leading to GC>TA transversion mutations.
Project description:The formation of oxidative lesions arising from double stranded DNA damage is of major significance to chemical biology from the perspective of application to human health. The quantification of purine lesions arising from ?-radiation-induced hydroxyl radicals (HO(•)) has been the subject of numerous studies, with discrepancies on the measured 5',8-cyclo-2'-deoxyadenosine (cdA) and 5',8-cyclo-2'-deoxyguanosine (cdG) lesions reported by different groups. Here we applied an ameliorative protocol for the analysis of DNA damage with quantitative determination of these lesions via isotope dilution liquid chromatography coupled with tandem mass spectrometry. Tandem-type purine lesions were quantified along with 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) and 7,8-dihydro-8-oxo-2'-deoxyadenosine (8-oxo-dA) in single and double stranded DNA, generated during DNA exposure to diffusible HO(•) radicals in the absence or presence of physiological levels of oxygen. The cdA and cdG lesions in absence of oxygen were found ~2 times higher in single than double stranded DNA, with 5'R being ~6.5 and ~1.5 times more predominant than 5'S in cdG and cdA, respectively. Interestingly, in the presence of 5% molecular oxygen the R/S ratios are retained with substantially decreased yields for cdA and cdG, whereas 8-oxo-dA and 8-oxo-dG remain nearly constant. The overall lesion formation follows the order: 8-oxo-dG >> 8-oxo-dA > 5'R-cdG > 5'R-cdA > 5'S-cdA > 5'S-cdG. By this method, there was a conclusive evaluation of radiation-induced DNA purine lesions.
Project description:A major DNA oxidation product, 2,2-diamino-4-[(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-5(2H)-oxazolone (oxazolone), can be generated either directly by oxidation of dG or as a secondary oxidation product with an intermediate of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG). Site-specific mutagenesis studies indicate that oxazolone is a strongly mispairing lesion, inducing approximately 10-fold more mutations than 8-oxo-dG. While 8-oxo-dG undergoes facile further oxidation, oxazolone appears to be a stable final product of guanine oxidation, and, if formed in vivo, can potentially serve as a biomarker of DNA damage induced by oxidative stress. In this study, capillary liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) methods were developed to enable quantitative analysis of both 8-oxo-dG and oxazolone in DNA from biological sources. Sensitive and specific detection of 8-oxo-dG and oxazolone in enzymatic DNA hydrolysates was achieved by isotope dilution with the corresponding 15N-labeled internal standards. Both nucleobase adducts were formed in a dose-dependent manner in calf thymus DNA subjected to photooxidation in the presence of riboflavin. While the amounts of oxazolone continued to increase with the duration of irradiation, those of 8-oxo-dG reached a maximum at 20 min, suggesting that 8-oxo-dG is converted to secondary oxidation products. Both lesions were found in rat liver DNA isolated under carefully monitored conditions to minimize artifactual oxidation. Liver DNA of diabetic and control rats maintained on a diet high in animal fat contained 2-6 molecules of oxazolone per 10(7) guanines, while 8-oxo-dG amounts in the same samples were between 3 and 8 adducts per 10(6) guanines. The formation of oxazolone lesions in rat liver DNA, their relative stability in the presence of oxidants and their potent mispairing characteristics suggest that oxazolone may play a role in oxidative stress-mediated mutagenesis.
Project description:Y-family DNA polymerases are known to bypass DNA lesions in vitro and in vivo and rescue stalled DNA replication machinery. Dpo4, a well-characterized model Y-family DNA polymerase, is known to catalyze translesion synthesis across a variety of DNA lesions including 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxo-dG). Our previous X-ray crystallographic, stopped-flow Förster resonance energy transfer (FRET), and computational simulation studies have revealed that Dpo4 samples a variety of global conformations as it recognizes and binds DNA. Here we employed single-molecule FRET (smFRET) techniques to investigate the kinetics and conformational dynamics of Dpo4 when it encountered 8-oxo-dG, a major oxidative lesion with high mutagenic potential. Our smFRET data indicated that Dpo4 bound the DNA substrate in multiple conformations, as suggested by three observed FRET states. An incoming correct or incorrect nucleotide affected the distribution and stability of these states with the correct nucleotide completely shifting the equilibrium toward a catalytically competent complex. Furthermore, the presence of the 8-oxo-dG lesion in the DNA stabilized both the binary and ternary complexes of Dpo4. Thus, our smFRET analysis provided a basis for the enhanced efficiency which Dpo4 is known to exhibit when replicating across from 8-oxo-dG.
Project description:Cellular DNA is constantly exposed to oxidative stress from both exogenous and endogenous sources, creating lesions that lead to aging related diseases, including cancer. 8-Oxo-guanine (8OG) is one of the most common forms of oxidative DNA damage, and failure to repair this lesion results in G:C to T:A transversion. Another common lesion, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapydG), shares the same precursor as 8OG. In Escherichia coli, both lesions are recognized and excised by the DNA glycosylase Fpg. X-ray crystallographic studies have shown that FapydG and 8OG adopt different conformations in the active site of Fpg. Our simulations suggest that the different binding modes observed for 8OG and FapydG arise directly from response to the nonconserved E77 present in the thermophilic Fpg sequences used for the crystallography experiments. In simulations with consensus S77, these lesions adopt very similar binding modes.
Project description:The oral pathogen, Streptococcus mutans, possesses inducible DNA repair defences for protection against pH fluctuations and production of reactive oxygen metabolites such as hydrogen peroxide (H(2) O(2) ), which are present in the oral cavity. DNA base excision repair (BER) has a critical role in genome maintenance by preventing the accumulation of mutations associated with environmental factors and normal products of cellular metabolism. In this study, we examined the consequences of compromising the DNA glycosylases (Fpg and MutY) and endonucleases (Smx and Smn) of the BER pathway and their relative role in adaptation and virulence. Enzymatic characterization of the BER system showed that it protects the organism against the effects of the highly mutagenic lesion, 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxo-dG). S. mutans strains lacking a functional Fpg, MutY or Smn showed elevated spontaneous mutation frequencies; and, these mutator phenotypes correlated with the ability of the strains to survive killing by acid and oxidative agents. In addition, in the Galleria mellonella virulence model, strains of S. mutans deficient in Fpg, MutY and Smn showed increased virulence as compared with the parent strain. Our results suggest that, for S. mutans, mutator phenotypes, due to loss of BER enzymes, may confer an advantage to virulence of the organism.
Project description:Polychlorinated biphenyls (PCBs) are organic chemicals that were traditionally produced and widely used in industry as mixtures and are presently formed as byproducts of pigment and dye manufacturing. They are known to persist and bioaccumulate in the environment. Some have been shown to induce liver cancer in rodents. Although the mechanism of the toxicity of PCBs is unknown, it has been shown that they increase oxidative stress, including lipid peroxidation. We hypothesized that oxidative stress-induced DNA damage could be a contributor for PCB carcinogenesis and analyzed several DNA adducts in female Sprague-Dawley rats exposed to 3,3',4,4',5-pentachlorobiphenyl (PCB 126), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153), and a binary mixture (PCB 126 + 153) for 14, 31, and 53 wks. Eight adducts were measured to profile oxidative DNA lesions, including 8-oxo-deoxyguanosine (8-oxo-dG), 1,N(6)-ethenodeoxyadenosine (1,N(6)-εdA), N(2),3-ethenoguanine (N(2),3-εG), 1,N(2)-ethenodeoxyguanosine (1,N(2)-εdG), as well as malondialdehyde (M1dG), acrolein (AcrdG), crotonaldehyde (CrdG), and 4-hydroxynonenal-derived dG adducts (HNEdG) by LC-MS/MS analysis. Statistically significant increases were observed for 8-oxo-dG and 1,N(6)-εdA concentrations in hepatic DNA of female rats exposed to the binary mixture (1000 ng/kg/day + 1000 μg/kg/day) but not in rats exposed to PCB 126 (1000 ng/kg/day) or PCB 153 (1000 μg/kg/day) for 14 and 31 wks. However, exposure to PCB 126 (1000 ng/kg/day) for 53 wks significantly increased 8-oxo-dG, 1,N(6)-εdA, AcrdG, and M1dG. Exposure to PCB 153 (1000 μg/kg/day) for 53 wks increased 8-oxo-dG, and 1,N(6)-εdA. Exposure to the binary mixture for 53 wks increased 8-oxo-dG, 1,N(6)-εdA, AcrdG, 1,N(2)-εdG, and N(2),3-εG significantly above control groups. Increased hepatic oxidative DNA adducts following exposure to PCB 126, PCB 153, or the binary mixture shows that an increase in DNA damage may play an important role in hepatic toxicity and carcinogenesis in female Sprague-Dawley rats.