Pyrimidine DNA Research Articles

A novel global genome method to measure and map DNA damage: application for chemotherapy treatment stratification

BackgroundMany chemotherapy agents act by inducing DNA damage, leading to cell death. Resistance and treatment failure limits the efficacy of these agents, and repair of the DNA damage is the commonest cause of resistance. By combining DNA immunoprecipitation with DNA microarray technology, we have developed a novel method to detect, quantify, and localise DNA damage at high resolution throughout the human genome. MethodsWe developed this assay initially in the model yeast organism Saccharomyces cerevisiae, before further development in human dermal fibroblast cells. Cellular DNA was treated in vivo or naked DNA treated in vitro with cisplatin, oxaliplatin, or ultraviolet (UV) irradiation. Using antibodies against cisplatin-modified DNA or cyclobutane pyrimidine dimers, we captured damaged DNA fragments through DNA immunoprecipitation. DNA samples were then amplified, labelled and hybridised to custom-designed DNA microarrays to generate high-resolution genome-wide profiles for DNA damage. FindingsGenome-wide cisplatin, oxaliplatin, and UV-induced damage profiles were generated in human cells and were consistent, reproducible, and correlated well with mathematical models predicting sites of damage induction developed in our laboratory (r=0·7–0·8). Using standard chromatin immunoprecipitation-DNA microarray experiments, we generated genome-wide epigenetic profiles showing increased H3K14 acetylation after platinum exposure. By applying novel bioinformatic outlier detection methods, we identified genomic regions where differences occured between in-vitro and in-vivo damage profiles, and we demonstrated how this information could be integrated and correlated with epigenetic changes. InterpretationWe validated a novel technique using DNA microarrays to sensitively measure DNA damage and repair at high resolution throughout the human genome, and we used the platinum analogue chemotherapy agents and UV irradiation as a model for damage induction. This technology can be applied to other DNA damaging agents and to clinical samples taken from patients after treatment. We believe that by applying this assay, and using our bioinformatic approaches for integrating genomic and epigenetic datasets, we will be able to detect functional biomarkers and genetic signatures predictive of response or toxicity, allowing us to optimise and stratify the use of current therapies. We are applying these assays to clinical samples. FundingUK Medical Research Council, Velindre NHS Trust Charitable Funds.

Electron transport through 5-substituted pyrimidines in DNA: electron affinities of uracil and cytosine derivatives differently affect the apparent efficiencies

We investigated excess electron transport (EET) in DNA containing cytosine derivatives. By arranging the derivatives according to their electron affinities, the apparent EET efficiency was successfully regulated. Unexpectedly, however, providing gradients of electron affinity by inserting 5-fluorocytosine did not always enhance EET.

The significance of folate metabolism in complications of pregnant women

Proper metabolism of folates has a crucial role for body homeostasis. Folate metabolism regulates changing of amino acids (homocysteine and methionine), purine and pyrimidine synthesis and DNA methylation. These whole biochemical processes have significant influence on hematopoietic, cardiovascular and nervous system functions. The disturbances of folate cycle could result in chronic hypertension, coronary artery disease, higher risk of heart infarction, could promote cancers development, and psychic and neurodegenerative diseases. No less important is the connection with complications appearing in pregnant woman (recurrent miscarriages, preeclampsia, fetus hypotrophy intrauterine death, preterm placenta ablation, preterm delivery) and fetus defects (Down syndrome, spina bifida, encephalomeningocele, myelomeningocele). The complex process of folate metabolism requires adequate activity of many enzymes and presence of co-enzymes. A key enzyme in folate metabolism is methylenetetrahydrofolate reductase (MTHFR - methylenetetrahydrofolate reductase), and 677C>T polymorphism of MTHFR gene is connected with lower enzymatic activity In several researches it was indicated that 677C>T MTHFR polymorphism is an independent factor influencing homocysteine concentration in serum, and also folate concentration in serum and red blood cells. Nevertheless, it was also observed the correlation of 677C>T MTHFR polymorphism with Down syndrome, and neural tube defects appearance in fetus. In European populations frequency of mutated 677TT genotype ranges from a few to several percent. Women carriers of 677TT or 677CT MTHFR genotypes are exposed on folate metabolism disturbances and on the consequences of incorrect folate process during pregnancy Nowadays in this group of women folic acid supplementation is widely recommended. In the light of modern knowledge the attention was also focused on the importance of metafolin administration that omitted pathways of folic acid transformation after administration, and in pregnant women certainly is valuable complement of supplementation in this respect.

Electrochemical determination of purine and pyrimidine DNA bases based on the recognition properties of azocalix[4]arene

The azocalix[4]arene film modified glassy carbon electrode was established for the convenient and sensitive detection of four DNA bases (guanine, adenine, thymine and cytosine). Field emission scanning electron microscopy, attenuated total reflectance-FTIR and X-ray photoelectron spectroscopy were used to characterize the film. The azocalix[4]arene film exhibited excellent electrocatalytic activity toward the oxidation of all bases. Well-separated voltammetric peaks were obtained among guanine, adenine, thymine and cytosine, which lead to the feasibility for the simultaneous determination of all of them in a mixture without separation or pretreatment. Linear calibration curves were obtained from 0.125 to 200.0μM for adenine, 0.125 to 175.0μM for guanine, 2.50 to 650.0μM for thymine, and 2.50 to 650.0μM for cytosine. This sensor also exhibits good stability, reproducibility and long lifetime.

R-matrix study of elastic and inelastic electron collisions with cytosine and thymine

Ab initio scattering calculations for low-energy electron collisions with gas-phase DNA pyrimidine bases (cytosine and thymine) are performed using the R-matrix method. The low-lying resonant states of the anions, formed due to electron capture by the bases, are identified using different levels of theory, including static exchange, static exchange plus polarization, and close-coupling approximations. For both the bases, all of our scattering models find three shape resonances of 2A″ (π) symmetry lying below 10 eV, in accordance with other calculations and experimental observations. In addition to these, a fourth 2A″ resonance is found in all models for thymine and in our best model, i.e. the uncontracted close-coupling approximation, for cytosine. This model places the fourth 2A″ resonance in both systems in the 6–7 eV range. No evidence is found for the presence of low-lying Feshbach resonances in either system. Integral cross sections for electronically elastic and inelastic collisions are also presented.

The Cyclobutane Dimers of 2′‐Deoxyuridine, 2′‐Deoxycytidine, 5‐Methyl‐2′‐Deoxycytidine and 5‐Bromo‐2′‐Deoxyuridine

Irradiation of DNA and RNA pyrimidine nucleosides with UV light in frozen aqueous solution or in solution with acetone often results in the formation of cyclobutane dimers (CBDs). Many of these photodimers have not been characterized. We present here the results of work designed to achieve the isolation, spectroscopic characterization and determination of the stereochemical nature of a number of little studied or previously unstudied CBDs of four 2'-deoxyribonuclesides. These nucleosides are 2'-deoxyuridine (dUrd), 2'-deoxycytidine (dCyd), 5-methyl-2'-deoxycytidine (5-MedCyd) and 5-bromo-2'-deoxyuridine (5-BrdUrd). In particular, we have isolated and characterized six dUrd CBDs, five dCyd CBDs, five 5-MedCyd CBDs and four 5-BrdUrd CBDs. Photoproducts were studied by UV spectroscopy, mass spectrometry, proton NMR spectroscopy and via chemical approaches. Also presented are results from less definitive studies of a number of (6-4) (or 5-4) photoadducts of these nucleosides. In addition, results from exploratory photochemical studies of other 2'-deoxyribonucleosides in frozen solution, as well as some mixtures of two nucleosides, are given. The latter results indicate that 5-iodo-2'-deoxyuridine (5-IdUrd), 5-bromo-2'-deoxycytidine and 5-iodo-2'-deoxycytidine each form putative CBDs and that 5-BrdUrd is capable of forming putative mixed CBDs and (6-4) and/or (5-4) adducts with thymidine (Thd); 5-IdUrd similarly forms a (6-4) (or (5-4)) adduct with Thd.

Vitamins at physiological levels cause oxidation to the DNA nucleoside deoxyguanosine and to DNA--alone or in synergism with metals

Vitamins with antioxidant properties have the ability to act as pro-oxidants, inducing oxidative damage and oxidative stress as opposed to preventing it. While vitamin supplements are commonly consumed, the scientific evidence for their health beneficial effects is inconclusive. In fact, even harmful effects have been reported. The present study aimed to investigate and compare pro-oxidant properties of different antioxidants and vitamins commonly found in dietary supplements, at concentrations of physiological relevance, alone or in combination with metals also found in supplements. Focus was on damages related to DNA. The vitamins' chemical oxidation potencies were studied by measuring the amount of the oxidation product 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formed from the DNA nucleoside deoxyguanosine (dG) after vitamin exposure, using a high-performance liquid chromatography system with electrochemical and ultraviolet detection. To study the vitamins' ability to cause DNA damage to cultured cells, promyelocytic leukemia cells (HL-60) were exposed to vitamins, and strand breaks, alkali-labile sites and oxidative DNA lesions, i.e. formamido pyrimidine DNA glycosylase-sensitive sites, were detected using the comet assay. Vitamins A and C chemically induced oxidation of dG, alone and in synergism with iron or copper, whereas only vitamin C and copper induced DNA damage in cultured cells. Contrary, vitamins B1, B2, B3, B6 and B12, β-carotene, folic acid, α-tocopherol, δ-tocopherol or γ-tocopherol did not induce oxidative damage to dG, while lycopene induced a weak dose-response increase. Taken together, vitamin C and copper stood out with the strongest oxidative potency, which is of potential concern since both substances are commonly found in multivitamins.

Extracellular Recombinant Annexin II Confers UVC-Radiation Resistance and Increases the Bcl-xL to Bax Protein Ratios in Human UVC-Radiation-Sensitive Cells

In this study, we found that refractoriness to ultraviolet (UVC) light-induced cell death was increased in UVC-radiation-sensitive cells derived from Cockayne syndrome patients when the cells were precultured in medium supplemented with recombinant annexin II (rANX II). In CS3BES cells, an immortal cell line derived from Cockayne syndrome patients, the rANX II supplementation-induced UVC-radiation resistance was suppressed by treatment with an anti-annexin II antibody and EGTA. The amount of biotinylated annexin II on the cell surface increased in the rANX II-supplemented cells but did not increase in the cells that were cotreated with rANX II and EGTA. The capacity to remove UVC-radiation-damaged DNA, (6-4) photoproducts and cyclobutane pyrimidine dimers, was the same in cells that were precultured with rANX II and in control cells that did not receive rANX II supplementation. The rANX II supplementation-induced UVC-radiation resistance was also observed in nucleotide excision repair-deficient cells and xeroderma pigmentosum group A-downregulated cells. The Bcl-xL to Bax protein ratios, an index of survival activity in cells exposed to lethal stresses, were increased in the cells that had been precultured in rANX II for 24 h prior to UVC irradiation. Treatment with a phosphatidylinositol 3-kinase inhibitor suppressed the increased UVC-radiation resistance and Bcl-xL to Bax ratios in the cells with rANX II supplementation. Furthermore, downregulation of Bcl-xL by siRNA transfection also suppressed the UVC-radiation resistance that was induced by rANX II supplementation. These results suggest that the increase in the Bcl-xL to Bax ratios may be associated with enhanced resistance to UVC-radiation-induced cell death.

Interactions between enhancer of rudimentary and Notch and deltex reveal a regulatory function of enhancer of rudimentary in the Notch signaling pathway in Drosophila melanogaster

Enhancer of rudimentary, e(r), encodes a small nuclear protein, ER, that has been implicated in the regulation of pyrimidine metabolism, DNA replication, and cell proliferation. In Drosophila melanogaster, a new recessive Notch allele, Nnd-p, was isolated as a lethal in combination with an e(r) allele, e(r)p2. Both mutants are viable as single mutants. Nnd-p is caused by a P-element insertion in the 5' UTR, 378-bp upstream of the start of translation. Together the molecular and genetic data argue that Nnd-p is a hypomorphic allele of N. The three viable notchoid alleles, Nnd-p, Nnd-1, and Nnd-3, are lethal in combination with e(r)- alleles. Our present hypothesis is that e(r) is a positive regulator of the Notch signaling pathway and that the lethality of the Ne(r) double mutants is caused by a reduction in the expression of the pathway. This is supported by the rescue of the lethality by a mutation in Hairless, a negative regulator of N, and by the synthetic lethality of dxe(r) double mutants. Further support for the hypothesis is a reduction in E(spl) expression in an e(r)- mutant. Immunostaining localizes ER to the nucleus, suggesting a nuclear function for ER. A role in the Notch signaling pathway, suggests that e(r) may be expressed in the nervous system. This turns out to be the case, as immunostaining of ER shows that ER is localized to the developing CNS.

Mass-Tuned Initial Excited-State Structural Dynamics of DNA Nucleobases from UV Resonance Raman Spectroscopy: 5-Deuterouracil

5-Deuterouracil (5-d-U) is an analogue of uracil (RNA) and thymine (DNA), which differ only in the mass of their 5-substituent. Previous work has shown that the initial excited-state structural dynamics primarily lie along C5═C6 bond lengthening in thymine, while these dynamics primarily lie along C5 and C6 pyramidalization in uracil. To test whether the 5-substituent mass determines these dynamics, the resonance Raman spectra of 5-d-U, with a 5-substituent mass intermediate between uracil and thymine, is measured at wavelengths throughout its 262 nm absorption band. The resulting spectrum of 5-d-U is intermediate between that of thymine and uracil, indicating both bond-lengthening and pyramidalization motions in the excited state. Self-consistent analysis of the resonance Raman excitation profiles and absorption spectra show that the initial excited-state structural dynamics of 5-d-U are indeed intermediate between those of thymine and uracil, supporting the mass-tuning model of initial excited-state structural dynamics in DNA and RNA pyrimidine nucleobases.

Vitamin A and C compounds permitted in supplements differ in their abilities to affect cell viability, DNA and the DNA nucleoside deoxyguanosine

In accordance with the European Parliament and Council's directive, vitamin A and C supplements can include any of four (vitamin A) or five (vitamin C) specified compounds. This study focuses on these compounds and compares their abilities to affect the DNA and viability of cells in culture, but also their potencies to chemically oxidise the DNA nucleoside deoxyguanosine (dG). To study the vitamins' strict chemical oxidation potencies, dG was exposed to vitamin solution and the amount of the oxidation product 8'-hydroxydeoxyguanosine (8-oxodG) formed was estimated using a high-performance liquid chromatography system with electrochemical and ultraviolet detection. The vitamin's ability to cause DNA damage to promyelocytic leukaemia cells (HL-60), as detected by strand breaks, alkaline labile sites and formamido pyrimidine DNA glycosylase (FPG)-sensitive sites was, after vitamin exposure, measured using the comet assay and cytotoxicity was estimated using trypan blue staining. The results highlight that vitamin A and C compounds found in supplements do have different properties, chemically as well as in a cellular system. Among the vitamin C compounds, ascorbic acid, sodium ascorbate and calcium ascorbate stood out causing both oxidation to dG and cytotoxicity to cells. The vitamin A compounds retinol, retinyl acetate and retinal (a breakdown product found in vivo) caused oxidation of dG, while retinal was the only compound causing cytotoxicity, giving rise to an almost complete cell death. β-carotene caused, as the only vitamin compound, a small increase in FPG-sensitive sites. It is concluded that even though the compounds are found under the same name (vitamin A or C), they do have different properties linked to oxidation, cytotoxicity and DNA damage.

Kinetics of cyclobutane thymine dimer splitting by DNA photolyase directly monitored in the UV

CPD photolyase uses light to repair cyclobutane pyrimidine dimers (CPDs) formed between adjacent pyrimidines in UV-irradiated DNA. The enzyme harbors an FAD cofactor in fully reduced state (FADH(-)). The CPD repair mechanism involves electron transfer from photoexcited FADH(-) to the CPD, splitting of its intradimer bonds, and electron return to restore catalytically active FADH(-). The two electron transfer processes occur on time scales of 10(-10) and 10(-9)s, respectively. Until now, CPD splitting itself has only been poorly characterized by experiments. Using a previously unreported transient absorption setup, we succeeded in monitoring cyclobutane thymine dimer repair in the main UV absorption band of intact thymine at 266nm. Flavin transitions that overlay DNA-based absorption changes at 266nm were monitored independently in the visible and subtracted to obtain the true repair kinetics. Restoration of intact thymine showed a short lag and a biexponential rise with time constants of 0.2 and 1.5ns. We assign these two time constants to splitting of the intradimer bonds (creating one intact thymine and one thymine anion radical T(∘-)) and electron return from T(∘-) to the FAD cofactor with recovery of the second thymine, respectively. Previous model studies and computer simulations yielded various CPD splitting times between <1ps and <100ns. Our experimental results should serve as a benchmark for future efforts to model enzymatic photorepair. The technique and methods developed here may be applied to monitor other photoreactions involving DNA.

Electron paramagnetic resonance study of unpaired electron species in thin films of pyrimidine bases induced by nitrogen and oxygen K-shell photoabsorption

In order to clarify the mechanism of DNA-base modification induced by K-shell photoabsorption of nitrogen and oxygen atoms, we measured electron paramagnetic resonance (EPR) spectra of two DNA pyrimidine bases, thymine, and cytosine. The g-factor of 2.000 of the unpaired electron species arising only during irradiation is determined. The EPR intensities for cytosine are two times larger than those simply estimated based on the photoabsorption cross section, whereas those for thymine show similar energy dependence to photoabsorption spectra, suggesting that cytosine favors to form unpaired electron species, rather than thymine, presumably due to the excitation of the enhanced electron capturing.

SiC nanoparticles-modified glassy carbon electrodes for simultaneous determination of purine and pyrimidine DNA bases

For the first time a novel and simple electrochemical method was used for simultaneous detection of DNA bases (guanine, adenine, thymine and cytosine) without any pretreatment or separation process. Glassy carbon electrode modified with silicon carbide nanoparticles (SiCNP/GC), have been used for electrocatalytic oxidation of purine (guanine and adenine) and pyrimidine bases (thymine and cytosine) nucleotides. Field emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) techniques were used to examine the structure of the SiCNP/GC modified electrode. The modified electrode shows excellent electrocatalytic activity toward guanine, adenine, thymine and cytosine. Differential pulse voltammetry (DPV) was proposed for simultaneous determination of four DNA bases. The effects of different parameters such as the thickness of SiC layer, pulse amplitude, scan rate, supporting electrolyte composition and pH were optimized to obtain the best peak potential separation and higher sensitivity. Detection limit, sensitivity and linear concentration range of the modified electrode toward proposed analytes were calculated for, guanine, adenine, thymine and cytosine, respectively. As shown this sensor can be used for nanomolar or micromolar detection of different DNA bases simultaneously or individually. This sensor also exhibits good stability, reproducibility and long lifetime.

DNA (6−4) Photolesion Repair Occurs in the Electronic Ground State of the TT Dinucleotide Dimer Radical Anion

(6−4) Photolyases are enzymes utilizing light to repair DNA pyrimidine−pyrimidone photolesions. The first step of enzyme function involves absorption of a photon and electron transfer from a flavin adenine dinucleotide anion (FADH−) to the (6−4) lesion-inducing repair, that is, splitting of the covalently bound nucleobase dimer. Here we demonstrate that the repair mechanism occurs in the electronic ground state of the lesion radical anion because the initially absorbed photon energy is not sufficient to initiate electron transfer and to excite electronically the radical anion of the (6−4) lesion simultaneously.

The role of oxidative DNA damage, DNA repair, GSTM1, SOD2 and OGG1 polymorphisms in individual susceptibility to Barrett's esophagus

Determination of the genetic alterations, which play a role in the etiology of Barrett’s esophagus (BE), could help identify high-risk individuals for esophageal adenocarcinoma (EA). The aim of the present study was to investigate the role of oxidative DNA damage, glutathione (GSH) concentration as oxidative stress parameters and DNA repair capacity, GSTM1, SOD1 Ala16Val and OGG1 Ser326Cys genetic polymorphisms as individual susceptibility parameters in the etiology of BE. The study groups comprised BE patients who were clinically diagnosed (n = 40) and a healthy control group (n = 40). Basal DNA damage, pyrimidine and purine base damage after H2O2 induction, H 2O2 sensitivity, DNA repair capacity, oxidized pyrimidine and purine base damage repair were evaluated in peripheral blood lymphocytes with a modified comet assay using specific endonucleases (Endo III and Fpg). Polymerase chain reaction—restriction length polymorphism (PCR-RFLP)-based assays were used for genotyping. The patient group showed eleva...

The role of oxidative DNA damage, DNA repair, GSTM1, SOD2 and OGG1 polymorphisms in individual susceptibility to Barrett’s esophagus

Determination of the genetic alterations, which play a role in the etiology of Barrett's esophagus (BE), could help identify high-risk individuals for esophageal adenocarcinoma (EA). The aim of the present study was to investigate the role of oxidative DNA damage, glutathione (GSH) concentration as oxidative stress parameters and DNA repair capacity, GSTM1, SOD1 Ala16Val and OGG1 Ser326Cys genetic polymorphisms as individual susceptibility parameters in the etiology of BE. The study groups comprised BE patients who were clinically diagnosed (n = 40) and a healthy control group (n = 40). Basal DNA damage, pyrimidine and purine base damage after H(2)O(2) induction, H( 2)O(2) sensitivity, DNA repair capacity, oxidized pyrimidine and purine base damage repair were evaluated in peripheral blood lymphocytes with a modified comet assay using specific endonucleases (Endo III and Fpg). Polymerase chain reaction-restriction length polymorphism (PCR-RFLP)-based assays were used for genotyping. The patient group showed elevated levels of basal DNA damage, pyrimidine base damage and H(2)O(2) sensitivity as compared to controls (p < .05). DNA repair capacity, oxidized pyrimidine and purine base damage repair capacity, were not statistically different between patients and controls. GSH concentration was found to be significantly lower in smoking patients than in the controls (p < .05). None of the genetic variations changed the risk of having BE disease. However, patients carrying the variant OGG1 Cys allele showed elevated levels of pyrimidine base damage as compared to patients carrying the wild-type OGG1 Ser (p < .05). The results of this study point to a role of oxidative DNA damage in BE. However, DNA repair capacity, GSTM1, SOD1 Ala16Val and OGG1 Ser326Cys genetic polymorphisms appeared to play no role in the individual susceptibility to this disease.

Sensitized formation of oxidatively generated damage to cellular DNA by UVA radiation

The survey is aimed at critically reviewing information on the UVA-mediated oxidative reactions to cellular components with emphasis on DNA as the result of mostly photosensitized pathways. It appears clearly that UVA radiation is relatively much more efficient than UVB photons in inducing oxidative processes. The main UVA-induced oxidative degradation pathways of DNA are reported and discussed mechanistically. They are mostly rationalized in terms of a major contribution of singlet molecular oxygen ((1)O(2)) and to a lesser extent of hydroxyl radical ( OH), that in the latter case originates from Fenton-type reactions. This leads to the predominant formation of 8-oxo-7,8-dihydroguanine together with smaller amounts of oxidized pyrimidine bases and DNA strand breaks in UVA-irradiated cells.

Proteomics of Porphyromonas gingivalis within a model oral microbial community

BackgroundPorphyromonas gingivalis is a periodontal pathogen that resides in a complex multispecies microbial biofilm community known as dental plaque. Confocal laser scanning microscopy showed that P. gingivalis can assemble into communities in vitro with Streptococcus gordonii and Fusobacterium nucleatum, common constituents of dental plaque. Whole cell quantitative proteomics, along with mutant construction and analysis, were conducted to investigate how P. gingivalis adapts to this three species community.Results1156 P. gingivalis proteins were detected qualitatively during comparison of the three species model community with P. gingivalis incubated alone under the same conditions. Integration of spectral counting and summed signal intensity analyses of the dataset showed that 403 proteins were down-regulated and 89 proteins up-regulated. The proteomics results were inspected manually and an ontology analysis conducted using DAVID. Significant decreases were seen in proteins involved in cell shape and the formation of the cell envelope, as well as thiamine, cobalamin, and pyrimidine synthesis and DNA repair. An overall increase was seen in proteins involved in protein synthesis. HmuR, a TonB dependent outer membrane receptor, was up-regulated in the community and an hmuR deficient mutant was deficient in three species community formation, but was unimpaired in its ability to form mono- or dual-species biofilms.ConclusionCollectively, these results indicate that P. gingivalis can assemble into a heterotypic community with F. nucleatum and S. gordonii, and that a community lifestyle provides physiologic support for P. gingivalis. Proteins such as HmuR, that are up-regulated, can be necessary for community structure.

Development of Tester Strains Deficient in Nth/Nei DNA Glycosylases to Selectively Detect the Mutagenicity of Oxidized DNA Pyrimidines

Oxidative DNA damage is a major cause of mutation and cell death in aerobic organisms. In addition to 8-hydroxyguanine, oxidized DNA pyrimidines play important roles in mutagenesis. Salmonella typhimurium TA1535, widely used in mutagenicity assays, carries a hisG46 missense mutation and efficiently detects mutations at G:C base pairs. To detect oxidative mutagens that selectively modify pyrimidines, we constructed a derivative of strain TA1535, termed YG3206, which lacks the Nei and Nth DNA glycosylases that excise oxidized pyrimidines from DNA. This novel strain easily detected the mutagenicity of L-cysteine, L-penicillamine, dopamine-HCl, and phenazine methosulfate, which are non-mutagenic or only weakly mutagenic in the TA1535 parent strain. A second strain that is equivalent to YG3206 but harbors the plasmid pKM101 which carries mucAB encoding DNA polymerase R1, termed YG3216, was significantly sensitive to phenazine ethosulfate. The compound was not mutagenic in either YG3206 or the Fapy-glycosylase-defective strain YG3001. Potassium bromate and methylene blue plus visible light with metabolic activation induced mutations in YG3001 but not YG3206 or YG3216. The number of spontaneous His+ revertants per plate was 82 ± 16 (YG3206, ΔnthΔnei), 19 ± 4 (YG3001, Δfpg), and 6 ± 2 (TA1535), suggesting a significant contribution to spontaneous mutagenesis by endogenous pyrimidine oxidation. In the absence of exogenous chemical treatment, exposure to fluorescent light enhanced the spontaneous mutation frequency by approximately two-fold (YG3206), 13-fold (YG3001), and 10-fold (TA1535). These results suggest that certain environmental chemicals may selectively introduce mutagenic damage at DNA pyrimidines, and that these changes can be monitored by the use of YG3206.