Repair of oxidative DNA damage

Cellular genomes suffer extensive damage from exogenous agents and reactive oxygen species formed during normal metabolism. The MutT homologs (MutT/MTH) remove oxidized nucleotide precursors so that they cannot be incorporated into DNA during replication. Among many repair pathways, the base excision repair (BER) pathway is the most important cellular protection mechanism responding to oxidative DNA damage. The 8-oxoG glycosylases (Fpg or MutM/OGG) and the MutY homologs (MutY/MYH) glycosylases along with MutT/MTH protect cells from the mutagenic effects of 8-oxoG, the most stable and deleterious product known caused by oxidative damage to DNA. The key enzymes in the BER process are DNA glycosylases, which remove different damaged bases by cleavage of the N-glycosylic bonds between the bases and the deoxyribose moieties of the nucleotide residues. Biochemical and structural studies have demonstrated the substrate recognition and reaction mechanism of BER enzymes. Cocrystal structures of strated the substrate recognition and reaction mechanism of BER enzymes. Cocrystal structures of several glycosylases show that the substrate base flips out of the sharply bent DNA helix and the minor groove is widened to be accessed by the glycosylases. To complete the repair after glycosylase action, the apurinic/apyrimidinic (AP) site is further processed by an incision step, DNA synthesis, an excision step, and DNA ligation through two alternative pathways. The short-patch BER (1-nucleotide patch size) and long-patch BER (2–6-nucleotide patch size) pathways need AP endonuclease to generate a 3′ hydroxyl group but require different sets of enzymes for DNA synthesis and ligation. Protein-protein interactions have been reported among the enzymes involved in BER. It is possible that the successive players in the repair pathway are assembled in a complex to perform concerted actions. The BER pathways are proposed to protect cells and organisms from mutagenesis and carcinogenesis.     

Repair and tolerance of oxidative DNA damage in plants

DNA damage caused by exposure to reactive oxygen species is one of the primary causes of DNA decay in most organisms. In plants, endogenous reactive oxygen species (ROS) are generated not only by respiration and photosynthesis, but also by active responses to certain environmental challenges, such as pathogen attack. Significant extracellular sources of activated oxygen include air pollutants such as ozone and oxidative effects of UV light and low-level ionizing radiation. Plants are well equipped to cope with oxidative damage to cellular macromolecules, including DNA. Oxidative attack on DNA generates both altered bases and damaged sugar residues that undergo fragmentation and lead to strand breaks. Recent advances in the study of DNA repair in higher plants show that they use mechanisms similar to those present in other eukaryotes to remove and/or tolerate oxidized bases and other oxidative DNA lesions. Therefore, plants represent a valuable model system for the study of DNA oxidative repair processes in eukaryotic cells.     

Repair of products of oxidative DNA base damage in human cells.

Oxidative DNA damage is the most frequent type of damage encountered by aerobic cells and may play an important role in biological processes such as mutagenesis, carcinogenesis and aging in humans. Oxidative damage generates a myriad of modifications in DNA. We investigated the cellular repair of DNA base damage products in DNA of cultured human lymphoblast cells, which were exposed to oxidative stress by H2O2. This DNA-damaging agent is known to cause base modifications in genomic DNA of mammalian cells [Dizdaroglu, M., Nackerdien, Z., Chao, B.-C., Gajewski, E. and Rao, G. (1991) Arch. Biochem. Biophys. 285, 388-390]. Following treatment with H2O2, the culture medium was freed from H2O2 and cells were incubated for time periods ranging from 10 min to 6 h. DNA was isolated from control cells, hydrogen peroxide-treated cells and cells incubated after H2O2 exposure. DNA samples were analyzed by gas chromatography/isotope-dilution mass spectrometry. Eleven modified bases were identified and quantified. The results showed a significant formation of these DNA base products upon H2O2-treatment of cells. Subsequent incubation of cells caused a time-dependent excision of these products from cellular DNA. The cell viability did not change significantly by various treatments. There were distinct differences between the kinetics of excision of individual products. The observed excisions were attributed to DNA repair in cells. The rate of repair of purine lesions was slower than that of pyrimidine lesions. Most of the identified products are known to possess various premutagenic properties. The results of this work may contribute to the understanding of the cellular repair of oxidative DNA damage in human and other mammalian cells.     

Base-excision repair of oxidative DNA damage by DNA glycosylases

Oxidative damage to DNA caused by free radicals and other oxidants generate base and sugar damage, strand breaks, clustered sites, tandem lesions and DNA-protein cross-links. Oxidative DNA damage is mainly repaired by base-excision repair in living cells with the involvement of DNA glycosylases in the first step and other enzymes in subsequent steps. DNA glycosylases remove modified bases from DNA, generating an apurinic/apyrimidinic (AP) site. Some of these enzymes that remove oxidatively modified DNA bases also possess AP-lyase activity to cleave DNA at AP sites. DNA glycosylases possess varying substrate specificities, and some of them exhibit cross-activity for removal of both pyrimidine- and purine-derived lesions. Most studies on substrate specificities and excision kinetics of DNA glycosylases were performed using oligonucleotides with a single modified base incorporated at a specific position. Other studies used high-molecular weight DNA containing multiple pyrimidine- and purine-derived lesions. In this case, substrate specificities and excision kinetics were found to be different from those observed with oligonucleotides. This paper reviews substrate specificities and excision kinetics of DNA glycosylases for removal of pyrimidine- and purine-derived lesions in high-molecular weight DNA.     

Repair of oxidative guanine damage in plasmid DNA by indoles involves proton transfer between complementary bases

We have used the single electron oxidizing agent (SCN)(2)(*)(-) (generated by gamma-irradiation of aqueous thiocyanate) to produce guanyl radicals in plasmid DNA. The stable product(s) formed from these radicals can be detected after conversion with a base excision repair endonuclease to single strand breaks. The yield of enzyme-induced breaks is decreased by the presence during irradiation of indole compounds. Rate constants for the reduction of DNA guanyl radicals by these indoles can be calculated from the concentration dependence of the attenuation in the yield of enzyme sensitive sites. Indoles bearing electron-donating groups (methoxy or methyl) appear to react at the diffusion-controlled rate, but those bearing electron-withdrawing groups (cyano or nitro) are significantly less reactive. At physiological pH values, the reduction of a DNA guanyl radical involves the transfer of a proton as well as an electron. Comparison of the kinetic results with literature thermodynamic data suggests that the source of this proton is the complementary base-paired cytosine.     

Repair of DNA O-alkylation damage by various human organs

Protein extracts from human adult liver, fetal liver, intestine, brain, kidney, lung and skin were tested against poly(dT)methylated X poly(dA), poly(dA)methylated X poly(dT) and methylated DNA. The suitability of various substrates was established in assays using E. coli extracts that removed O4-methylthymidine (O4-MedT), O2-MedT, and O6-methylguanine (O6-MeG). The human extracts efficiently removed O6-MeG and N3-methyladenine from methylated substrates. The adult liver exhibited low and fetal tissues negligible removal of O4-MedT. Only the liver showed limited removal of O2-MedT. The poor removal of the miscoding base O4-MedT by human organs could be an important factor in carcinogen induced mutagenesis, carcinogenesis and teratogenesis.     

Repair of N-methyl-N''-nitro-N-nitrosoguanidine-induced DNA damage by ABC excinuclease.

Escherichia coli has several overlapping DNA repair pathways which act in concert to eliminate the DNA damage caused by a diverse array of physical and chemical agents. The ABC excinuclease which is encoded by the uvrA, uvrB, and uvrC genes mediates both the incision and excision steps of nucleotide excision repair. Traditionally, this repair pathway has been assumed to be active against DNA adducts that cause major helical distortions. To determine the level of helical deformity required for recognition and repair by ABC excinuclease, we have evaluated the substrate specificity of this enzyme by using DNA damaged by N-methyl-N'-nitro-N-nitrosoguanidine. ABC excinuclease incised methylated DNA in vitro in a dose-dependent manner in a reaction that was ATP dependent and specific for the fully reconstituted enzyme. In vivo studies with various alkylation repair-deficient mutants indicated that the excinuclease participated in the repair of DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine.     

Repair of UV damage in plasmid DNA by human fibroblasts

Summary Plasmid DNA from Bacillus subtilis was introduced into monolayers of human fibroblasts by means of a modification of the calcium phosphate coprecipitation technique, comprising centrifugation of the coprecipitate onto the cells and treatment with polyethyleneglycol. The amount of DNA resistant to removal from the monolayers ranged from 10% to 15% of the input DNA. By determination of the biological activity of the plasmid DNA, re-extracted after various periods following entry into the fibroblasts and subsequently used as donor for B. subtilis protoplasts, it was shown that the activity of the plasmid DNA was gradually lost. When ultraviolet light-inactivated plasmid DNA was used as donor, reactivation of the plasmid was observed, which was completed within 2 h. The dose-dependent incorporation of [¹⁴C]-thymidine suggests that DNA repair processes were involved in reactivation of the plasmid DNA.     

Experimental study of oxidative DNA damage

Animal experiments allow the study of oxidative DNA damage in target organs and the elucidation of dose-response relationships of carcinogenic and other harmful chemicals and conditions as well as the study of interactions of several factors. So far the effects of more than 50 different chemical compounds have been studied in animal experiments mainly in rats and mice, and generally with measurement of 8-oxodG with HPLC-EC. A large number of well-known carcinogens induce 8-oxodG formation in liver and/or kidneys. Moreover several animal studies have shown a close relationship between induction of dative DNA damage and tumour formation.

In principle the level of oxidative DNA damage in an organ or cell may be studied by measurement of modified bases in extracted DNA by immunohistochemical visualisation, and from assays of strand breakage before and after treatment with repair enzymes. However, this level is a balance between the rates of damage and repair. Until the repair rates and capacity can be adequately assessed the rate of damage can only be estimated from the urinary excretion of repair products albeit only as an average of the entire body.

A number of model compounds have been used to induce oxidative DNA damage in experimental animals. The hepatocarcinogen 2-nitropropane induces up to 10-fold increases in 8-oxodG levels in rat liver DNA. The level of 8-oxodG is also increased in kidneys and bone marrow but not in the testis. By means of 2-nitropropane we have shown correspondence between the increases in 8-oxodG in target organs and the urinary excretion of 8-oxodG and between 8-oxodG formation and the comet assay in bone marrow as well potent preventive effects of extracts of Brussels sprouts. Others have shown similar effects of green tea extracts and its components. Drawbacks of the use of 2-nitropropane as a model for oxidative DNA damage relate particularly to formation of 8-aminoguanine derivatives that may interfere with HPLC-EC assays and have unknown consequences. Other model compounds for induction of oxidative DNA damage, such as ferric nitriloacetate, iron dextran, potassium bromate and paraquat, are less potent and/or more organ specific.

Inflammation and activation of an inflammatory response by phorbol esters or E. coli lipopolysaccharide (LPS) induce oxidative DNA damage in many target cells and enhance benzene-induced DNA damage in mouse bone marrow.

Experimental studies provide powerful tools to investigate agents inducing and preventing oxidative damage to DNA and its role in carcinogenesis. So far, most animal experiments have concerned 8-oxodG and determination of additional damaged bases should be employed. An ideal animal model for prevention of oxidative DNA damage has yet to he developed.     

Peptide repair of oxidative DNA damage

Guanyl radical species are produced in DNA by electron removal caused by ionizing radiation, photoionization, oxidation, or photosensitization. DNA guanyl radicals can be reduced by electron donation from mild reducing agents. Important biologically relevant examples are the redox active amino acids cysteine, cystine, methionine, tryptophan, and tyrosine. We have quantified the reactivity of derivatives of these amino acids with guanyl radicals located in plasmid DNA. The radicals were produced by electron removal using the single electron oxidizing agent (SCN)(2)(*)(-). Disulfides (cystine) are unreactive. Thioethers (methionine), thiols (cysteine), and phenols (tyrosine) react with rate constants in the range 10(4)-10(6), 10(5)-10(6), and 10(5)-10(6) dm(3) mol(-1) s(-1), respectively. Indoles (tryptophan) are the most reactive with rate constants of 10(7)-10(8) dm(3) mol(-1) s(-1). Selenium analogues of amino acids are over an order of magnitude more reactive than their sulfur equivalents. Increasing positive charge is associated with a ca. 10-fold increase in reactivity. The results suggest that amino acid residues located close to DNA (for example, in DNA binding proteins such as histones) might participate in the repair of oxidative DNA damage.     

Involvement of phylogenetically conserved acidic amino acid residues in catalysis by an oxidative DNA damage enzyme formamidopyrimidine glycosylase

Formamidopyrimidine glycosylase (Fpg) is an important bacterial base excision repair enzyme, which initiates removal of damaged purines such as the highly mutagenic 8-oxoguanine. Similar to other glycosylase/AP lyases, catalysis by Fpg is known to proceed by a nucleophilic attack by an amino group (the secondary amine of its N-terminal proline) on C1' of the deoxyribose sugar at a damaged base, which results in the departure of the base from the DNA and removal of the sugar ring by beta/delta-elimination. However, in contrast to other enzymes in this class, in which acidic amino acids have been shown to be essential for glycosyl and phosphodiester bond scission, the catalytically essential acidic residues have not been documented for Fpg. Multiple sequence alignments of conserved acidic residues in all known bacterial Fpg-like proteins revealed six conserved glutamic and aspartic acid residues. Site-directed mutagenesis was used to change glutamic and aspartic acid residues to glutamines and asparagines, respectively. While the Asp to Asn mutants had no effect on the incision activity on 8-oxoguanine-containing DNA, several of the substitutions at glutamates reduced Fpg activity on the 8-oxoguanosine DNA, with the E3Q and E174Q mutants being essentially devoid of activity. The AP lyase activity of all of the glutamic acid mutants was slightly reduced as compared to the wild-type enzyme. Sodium borohydride trapping of wild-type Fpg and its E3Q and E174Q mutants on 8-oxoguanosine or AP site containing DNA correlated with the relative activity of the mutants on either of these substrates.     

Repair of oxidative DNA damage in nuclear and mitochondrial DNA, and some changes with aging in mammalian cells

Exposure to exogenous and endogenous sources cause oxidative damage to cellular macromolecules, including DNA. This results in gradual accumulation of oxidative DNA base lesions, and in order to maintain genomic stability we must have effective systems to repair this kind of damage. The accumulation of lesions is most dramatic in the mitochondrial DNA, and this may cause dysfunction and loss of cellular energy production. Base excision DNA repair (BER) is the major pathway that removes oxidative DNA base lesions, and while we know much about its mechanism in the nuclear DNA, little is yet known about this pathway in mitochondria. While nuclear BER decreases with age, the mitochondrial DNA repair may increase with age. This increase is not enough to prevent the gradual accumulation of lesions in the mitochondrial DNA with age. Accumulation of DNA lesions with age may be the underlying cause for age-associated diseases including cancer.     

Repair of ochratoxin A-induced DNA damage and modulation of OTA-related genotoxicity by co-incubation with bile acids and methotrexatein vitro

The mycotoxin ochratoxin A (OTA) induced DNA strand breaks in porcine urinary bladder epithelial cells (PUBEC) and in Madin Darby canine kidney (MDCK) cells. A co-incubation with bile acids or methotrexate reduced or even prevented this adverse effect of OTAin vitro. The protective effect is possibly attributable to a decreased OTA uptake in cells, since bile acids and methotrexate are known to share common transport systems such as organic anion transporters (OAT) and/or organic anion transporting polypeptides (OATP) with the mycotoxin. OTA uptake in cells and its modulation can be one factor which determines the extent of adverse effects in different cell types. Another aspect of interest in this regard relates to repair of DNA damage: PUBEC cells are sensitive to OTA-induced damage which is more pronounced when DNA repair is blocked (by cytosine β-D-arabino-furanosid/hydroxyurea). On the other hand, when cells are kept in fresh (toxin-free) medium for 3 h, OTA-induced DNA damage decreased to control levels. Presented at the 26^th Mykotoxin-Workshop in Herrsching, Germany, May 17–19, 2004     

Mechanism of oxidative DNA damage induced by carcinogenic allyl isothiocyanate

Several isothiocyanates have been proposed as promising chemopreventive agents for human cancers. However, it has been reported that allyl isothiocyanate exhibit carcinogenic potential, and benzyl isothiocyanate and phenethyl isothiocyanate have tumor-promoting activities. We investigated whether these isothiocyanates could cause DNA damage, using (32)P-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. Allyl isothiocyanate caused Cu(II)-mediated DNA damage and formation of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG) more strongly than benzyl and phenethyl isothiocyanates. Catalase and bathocuproine, a Cu(I)-specific chelator, inhibited Cu(II)-mediated DNA damage by these isothiocyanates, suggesting involvement of H(2)O(2) and Cu(I). Isothiocyanates induced DNA damage frequently at thymine and cytosine residues in the presence of Cu(II). A UV-visible spectroscopic study revealed an association between the generation of superoxide and the yield of SH group from isothiocyanates. Furthermore, the yield of 8-oxodG formation was correlated with their superoxide-generating ability. Allyl isothiocyanate significantly induced 8-oxodG formation in HL-60 cells, but not in H(2)O(2)-resistant HP100 cells, suggesting the involvement of H(2)O(2) in cellular DNA damage. We conclude that oxidative DNA damage may play important roles in carcinogenic processes induced by allyl isothiocyanate.     

Mechanism of oxidative DNA damage induced by carcinogenic 4-aminobiphenyl

DNA adduct formation is thought to be a major cause of DNA damage by carcinogenic aromatic amines. We investigated the ability of an aromatic amine, 4-aminobiphenyl (4-ABP) and its N-hydroxy metabolite (4-ABP(NHOH)) to cause oxidative DNA damage, using (32)P-labeled human DNA fragments from the p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. 4-ABP(NHOH) was found to cause Cu(II)-mediated DNA damage, especially at thymine residues. Addition of the endogenous reductant NADH led to dramatic enhancement of this process. Catalase and bathocuproine, a Cu(I)-specific chelator, reduced the amount of DNA damage, suggesting the involvement of H(2)O(2) and Cu(I). 4-ABP(NHOH) dose-dependently induced 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation in the presence of Cu(ll) and NADH. 4-ABP(NHOH) conversion to nitrosobiphenyl, as measured by UV-visible spectroscopy, occurred rapidly in the presence of Cu(II), suggesting Cu(II)-mediated autoxidation. Increased amounts of 8-OHdG were found in HL-60 cells compared to the H(2)O(2)-resistant clone HP100 following 4-ABP(NHOH) treatment, further supporting the involvement of H(2)O(2). The present study demonstrates that an N-hydroxy derivative of 4-ABP induces oxidative DNA damage through H(2)O(2) in both a cell-free system and in cultured human cells. We conclude that, in addition to DNA adduct formation, oxidative DNA damage may play an important role in the carcinogenic process of 4-ABP.