Defective repair of oxidative damage in mitochondrial DNA in Down''s syndrome

BACKGROUND: Synthetic homopyrimidine peptide nucleic acids (PNAs) can bind complementary targets in double-stranded DNA, generating strand-displacement complexes, and so offering an opportunity to modulate specific gene expression. Several issues remain to be addressed before these attributes can be exploited in vivo, however. RESULTS: The kinetics of the interaction between a homopyrimidine PNA and a complementary homopurine target on double-stranded DNA were analyzed in the presence or absence of a preformed strand-displacement complex proximal to the target. The complex was established under low salt conditions by the binding of a different homopyrimidine PNA to a target situated adjacent to the first PNA target. These two targets were placed next to each other on opposite strands at distances of 0, 2, 4 and 8 base pairs apart. The presence of a preformed strand-displacement complex near the target accelerates the binding of PNA to double-stranded DNA in a salt-dependent manner. The influence of salt on the binding rates was also examined. The binding rate is increased by a factor of 1 x exp(70[NaCl]), that is, 16-fold at 40 mM NaCl and more than 10(4)-fold if extrapolated to 140 mM NaCl. This effect is significantly reduced if the two targets are 2 base pairs apart and completely absent if the distance is 4 base pairs or more. CONCLUSIONS: The perturbation of the DNA helix imposed by a PNA strand-displacement complex only propagates a few base pairs. It is therefore possible to target sites in the immediate vicinity of strand invasion complexes specifically. The results presented have implications for the mechanism of strand displacement and for the application of PNA in a genomic context.     

Mitochondrial targeting of human DNA glycosylases for repair of oxidative DNA damage

Oxidative damage to mitochondrial DNA has been implicated in human degenerative diseases and aging. Although removal of oxidative lesions from mitochondrial DNA occurs, the responsible DNA repair enzymes are poorly understood. By expressing the epitope-tagged proteins in COS-7 cells, we examined subcellular localizations of gene products of human DNA glycosylases: hOGG1, hMYH and hNTH1. A gene encoding for hOGG1 which excises 7,8-dihydro-8-oxoguanine (8-oxoG) from DNA generates four isoforms by alternative splicing (types 1a, 1b, 1c and 2). Three tagged isoforms (types 1b, 1c and 2) were localized in the mitochondria. Type 1a protein, which exclusively contains a putative nuclear localization signal, was sorted to the nucleus and lesser amount to the mitochondria. hMYH, a human homolog gene product of Escherichia coli mutY was mainly transported into the mitochondria. hNTH1 protein excising several pyrimidine lesions was transported into both the nucleus and mitochondria. In contrast to the three DNA glycosylases, translocation of the human major AP endonuclease (hAPE) into the mitochondria was hardly observed in COS-7 cells. These results suggest that the previously observed removal of oxidative base lesions in mitochondrial DNA is initiated by the above DNA glycosylases.     

Ultraviolet-induced DNA damage stimulates topoisomerase I-DNA complex formation in vivo: possible relationship with DNA repair

An antibody-based method was used to examine genomic DNA cleavage by endogenous topoisomerases in living cells. The method quantifies cleavable (covalent) complex formation in vivo after exposure to topoisomerase poisons, as reported previously (D. Subramanian et al., Cancer Res., 55: 2097-2103, 1995). Unexpectedly, exposing cells to UVB irradiation stimulated endogenous topoisomerase I-DNA covalent complex formation by as much as 8-fold, even in the absence of drugs that stabilize the cleavable complex. Covalent complexes are not a result of nonspecific UV protein-DNA cross-linking; rather, they result from the enzymatic activity of topoisomerase I on genomic DNA. Because the action of topoisomerase II on genomic DNA was not affected by UVB exposure, the observation appears to be specific for type I. Topoisomerase I is rapidly mobilized onto the genome (within 12 min after UVB exposure); however, topoisomerase I polypeptide levels did not show a corresponding increase, suggesting that preexisting enzyme is being recruited to sites of DNA damage. Complexes persist up to 5 h post-UV exposure (concurrent with the period of active DNA repair), and their formation is independent of S phase. These findings can be partially explained by the fact that in vitro topoisomerase I activity on UV-damaged DNA tends to favor formation of cleavage complexes; thus, a higher yield of covalent complexes are detected at or near cyclopyrimidine dimer lesions. Because repair-deficient cells are additionally compromised in their ability to recruit topoisomerase I, a direct role for the enzyme in DNA excision repair process in vivo is proposed that may be related to the activity of the xeroderma pigmentosum complementation group D helicase. Finally, these results collectively demonstrate that topoisomerase I is a repair-proficient topoisomerase in vivo.     

Mutagenicity and repair of oxidative DNA damage: insights from studies using defined lesions

Oxidative DNA damage has been implicated in mutagenesis, carcinogenesis and aging. Endogenous cellular processes such as aerobic metabolism generate reactive oxygen species (ROS) that interact with DNA to form dozens of DNA lesions. If unrepaired, these lesions can exert a number of deleterious effects including the induction of mutations. In an effort to understand the genetic consequences of cellular oxidative damage, many laboratories have determined the patterns of mutations generated by the interaction of ROS with DNA. Compilation of these mutational spectra has revealed that GC-->AT transitions and GC-->TA transversions are the most commonly observed mutations resulting from oxidative damage to DNA. Since mutational spectra convey only the end result of a complex cascade of events, which includes formation of multiple adducts, repair processing, and polymerase errors, it is difficult if not impossible to assess the mutational specificity of individual DNA lesions directly from these spectra. This problem is especially complicated in the case of oxidative DNA damage owing to the multiplicity of lesions formed by a single damaging agent. The task of assigning specific features of mutational spectra to individual DNA lesions has been made possible with the advent of a technology to analyze the mutational properties of single defined adducts, in vitro and in vivo. At the same time, parallel progress in the discovery and cloning of repair enzymes has advanced understanding of the biochemical mechanisms by which cells excise DNA damage. This combination of tools has brought our understanding of DNA lesions to a new level of sophistication. In this review, we summarize the known properties of individual oxidative lesions in terms of their structure, mutagenicity and repairability.     

DNA oxidative damage and life expectancy in houseflies

The objective of this study was to explore the relationship between oxidative molecular damage and the aging process by determining whether such damage is associated with the rate of aging, using the adult housefly as the experimental organism. Because the somatic tissues in the housefly consist of long-lived postmitotic cells, it provides an excellent model system for studying cumulative age-related cellular alterations. Rate of aging in the housefly was manipulated by varying the rate of metabolism (physical activity). The concentration of 8-hydroxydeoxyguanosine (80HdG) was used as an indicator of DNA oxidation. Exposure of live flies to x-rays and hyperoxia elevated the level of 8OHdG. The level of 8OHdG in mitochondrial as well as total DNA increased with the age of flies. Mitochondrial DNA was 3 times more susceptible to age-related oxidative damage than nuclear DNA. A decrease in the level of physical activity of the flies was found to prolong the life-span and corresponding reduce the level of 8OHdG in both mitochondrial and total DNA. Under all conditions examined, mitochondrial DNA exhibited a higher level of oxidative damage than total DNA. The 8OHdG levels were found to be inversely associated with the life expectancy of houseflies. The pattern of age-associated accrural of 8OHdG was virtually identical to that of protein carbonyl content. Altoghether, results of this study support the hypothesis that oxidative molecular damage is a causal factor in senescence.     

Epirubicin-induced oxidative DNA damage and evidence for its repair in lymphocytes of cancer patients who are undergoing chemotherapy

Anthracycline derivatives have been widely used in the treatment of several types of human malignancies. Cytotoxicity of these drugs has been attributed to inhibition of topoisomerase II as well as intracellular production of free radicals. In our work we used a gas chromatography/mass spectrometry technique to study free radical-induced DNA base modifications in chromatin isolated from lymphocytes of cancer patients who received chemotherapy with epirubicin (one of anthracycline's antitumor derivatives). The anticancer therapy caused significant increases in the amount of all four DNA base modifications over control levels in the lymphocytes of most of the patients. For the majority of the cases the base products returned to the control value 24 hr after the infusion of the drug, which suggests the removal of these lesions by cellular repair processes. However, some of the modified bases escaped repair. Because part of these modifications may possess premutagenic properties, they may be responsible for secondary cancers induced by chemotherapy.     

In vivo damage and recA-dependent repair of plasmid and chromosomal DNA in the radiation-resistant bacterium Deinococcus radiodurans

Deinococcus radiodurans R1 and other members of this genus share extraordinary resistance to the lethal and mutagenic effects of ionizing radiation. We have recently identified a RecA homolog in strain R1 and have shown that mutation of the corresponding gene causes marked radiosensitivity. We show here that following high-level exposure to gamma irradiation (1.75 megarads, the dose required to yield 37% of CFU for plateau-phase wild-type R1), the wild-type strain repairs > 150 double-strand breaks per chromosome, whereas a recA-defective mutant (rec30) repairs very few or none. A heterologous Escherichia coli-D. radiodurans shuttle plasmid (pMD68) was constructed and found to be retained in surviving D. radiodurans R1 and rec30 following any radiation exposure up to the highest dose tested, 3 megarads. Plasmid repair was monitored in vivo following irradiation with 1.75 megarads in both R1/pMD68 and rec30/pMD68. Immediately after irradiation, plasmids from both strains contained numerous breaks and failed to transform E. coli. While irradiation with 1.75 megarads was lethal to rec30 cultures, a small amount of supercoiled plasmid was regenerated, but it lacked the ability to transform E. coli. In contrast, wild-type cultures showed a cell division arrest of about 10 h, followed by exponential growth. Supercoiled plasmid was regenerated at normal levels, and it readily transformed E. coli. These studies show that D. radiodurans retains a heterologous plasmid following irradiation and repairs it with the same high efficiency as its chromosomal DNA, while the repair defect in rec30 prevents repair of the plasmid. Taken together, the results of this study suggest that plasmid DNA damaged in vivo in D. radiodurans is repaired by recA-dependent mechanisms similar to those employed in the repair of chromosomal DNA.     

Serum carotenoids and oxidative DNA damage in human lymphocytes

Carotenoids are thought to act as antioxidants in vivo, decreasing oxidative damage to biomolecules and thus protecting against coronary heart disease and cancer. However, human intervention studies with beta-carotene have given equivocal results in terms of cancer incidence. In an alternative molecular epidemiological approach, we have employed the 'comet assay' (single cell alkaline gel electrophoresis) to measure strand breaks, oxidized pyrimidines and altered purines in the DNA of lymphocytes from volunteers supplemented with alpha/beta-carotene, lutein, lycopene or placebo. In addition, we measured concentrations of the main serum carotenoids, and vitamins E and C, by HPLC. We report a significant negative correlation between basal concentrations of total serum carotenoids and oxidized pyrimidines. A similar correlation was seen between individual carotenoids (notably lutein and beta-carotene) and oxidized pyrimidines. However, carotenoid supplementation did not have a significant effect on endogenous oxidative damage. This suggests that there are some factors in the basal diet, probably found in fruit and vegetables, that decrease oxidative damage to DNA. In this case, basal serum carotenoids may simply be markers of consumption of fruit and vegetables, they themselves having little or no protective value.     

Comparison of oxidative base damage in mitochondrial and nuclear DNA

In 1994-1995, a child and five dogs from villages located between Jerusalem and Tel-Aviv, Israel were diagnosed with visceral leishmaniasis (VL). Based on these findings, the distribution of VL in domestic and wild canids in central Israel was examined. In the two villages where canine index cases were identified, a substantial proportion (11.5%, 14 of 122) of the dogs examined were seropositive. However, the rate of infection in five neighboring villages was only 1% (1 of 99). Parasites were cultured from 92% (12 of 13) of the seropositive dogs biopsied and the strains were characterized as Leishmania infantum by a clamped polymorphic-polymerase chain reaction, monoclonal antibodies, and/or excreted factor serology. The discovery of VL close to major urban centers is an important public health issue. The disease appears to have emerged recently in this area, and it is unclear whether the parasite was re-introduced or was continuously present at low levels in this region. The presence of seropositive wild canids, jackals (7.6%, 4 of 53) and red foxes (5%, 1 of 20), in central Israel, and the reappearance of the jackal population after near extinction suggests that wild canids may play a role in spreading this disease.     

Reversible induction of ATP synthesis by DNA damage and repair in Escherichia coli. In vivo NMR studies

Early metabolic events in Escherichia coli exposed to nalidixic acid, a topoisomerase II inhibitor and an inducer of the SOS system, were investigated by in vivo NMR spectroscopy, a technique that permits monitoring of bacteria under controlled physiological conditions. The energetics of AB1157 (wild type) and of its isogenic, SOS-defective mutants, recBC, lexA, and DeltarecA, were studied by 31P and 19F NMR before, during, and after exposure to nalidixic acid. The content of the NTP in E. coli embedded in agarose beads and perfused at 36 degreesC was found to be 4.3 +/- 1.1 x 10(-18) mol/cell, yielding a concentration of approximately 2.7 +/- 0.7 mM. Nalidixic acid induced in the wild type and mutants a rapid 2-fold increase in the content of the NTP, predominantly ATP. This induction did not involve synthesis of uracil derivatives or breakdown of RNA and caused cell proliferation to stop. Removal of nalidixic acid after 40 min of treatment rescued the cells and resulted in a decrease of ATP to control levels and resumption of proliferation. However, in DeltarecA cells, which were more sensitive to the activity of the drug, ATP elevation could not be reversed, and ATP content continued to increase faster than in control cells. The results ruled out association between the elevation of ATP and the induction of the SOS system and suggested involvement of a process reminiscent of apoptosis in the stimulation of ATP synthesis. Thus, the presence of the RecA protein was found to be essential for reversing the ATP increase and cell rescue, possibly by its function in repair of DNA damage.     

Haloacetate-induced oxidative damage to DNA in the liver of male B6C3F1 mice

Brominated and chlorinated haloacetates (HAs) are by-products of drinking water disinfection. Dichloroacetate (DCA) and trichloroacetate (TCA) are hepatocarcinogenic in rodents, but the brominated analogs have received little study. Prior work has indicated that acute doses of the brominated derivatives are more potent inducers of oxidative stress and increase the 8-hydroxydeoxyguanosine (8-OH-dG) content of the nuclear DNA in the liver. Since, DCA and TCA are also known as weak peroxisome proliferators, the present study was intended to determine whether this activity might be exacerbated by peroxisomal proliferation. Classical responses to peroxisome proliferators, cyanide-insensitive acyl-CoA oxidase activity and increased 12-hydroxylation of lauric acid, were elevated in a dose-related manner in mice maintained on TCA and clofibric acid (positive control), but not with DCA, dibromoacetate (DBA) or bromochloroacetate (BCA). Administration of the HAs in drinking water to male B6C3F1 mice for periods from 3 to 10 weeks resulted in dose-related increases in 8-OH-dG in nuclear DNA of the liver with DBA and BCA, but not with TCA or DCA. These findings indicate that oxidative damage induced by the haloacetates is, at least in part, independent of peroxisome proliferation. In addition, these data suggest that oxidative damage to DNA may play a more important role in the chronic toxicology of brominated compared to the chlorinated haloacetates.     

Mutagenicity of oxidative DNA damage in Chinese hamster V79 cells

We have investigated the mutagenicity of oxidative DNA damage induced in V79 Chinese hamster lung fibroblast, and measured 8-hydroxydeoxyguanosine (8OHdG) levels as an indicator of this damage. A hydroxyl radical generator, N,N'-bis(2-hydroxyperoxy-2-methoxyethyl)-1,4,5,8-naphthalene-tetra -carboxylic-diimide (NP-III), induced 8OHdG in V79 upon irradiation with 366 nm ultraviolet light (UV) for 15 min. 8OHdG was determined by HPLC with electrochemical detection after anaerobic sample processing. The 8OHdG level in the cells treated without NP-III was 0.49 per 10(5) dG, whereas levels in the cells treated with 5, 10 or 20 microM NP-III and UV irradiation were 1.84, 4.06 or 6.95 per 10(5) dG, respectively. The 8OHdG induced by 20 microM NP-III with UV irradiation decreased rapidly, and the half-life of the induced 8OHdG was approximately 6 h. NP-III with UV irradiation also induced DNA strand breaks in all cells uniformly, as determined by single cell gel assay. Mutant frequencies at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in V79 were determined as the number of 6-thioguanine-resistant cells per 10(6) cells. Mutant frequency of the cells without NP-III was 8.0, and frequencies of the cells treated with 5, 10 or 20 microM NP-III and UV irradiation were 14.9, 20.6 or 24.7 respectively. Treatment with 20 microM NP-III and UV irradiation decreased the cell number, determined 3 days after the treatment, to 20.8%. These findings indicate that acutely induced oxidative DNA damage including mutagenic 8OHdG is only weakly mutagenic in V79.     

Induction of oxidative DNA damage by ferric iron in mammalian cells

Ferric nitrilotriacetate (Fe-NTA) and ferric citrate (Fe-citrate) were compared with respect to their potential to induce oxidative DNA damage in V79 Chinese hamster cells. DNA base modifications, including 8-hydroxyguanine (7,8-dihydro-8-oxoguanine), were quantified by the frequency of lesions recognized by the bacterial Fpg protein (formamidopyrimidine-DNA glycosylase) in combination with the alkaline unwinding assay. Fe-NTA induced oxidative DNA damage in a time- and dose-dependent manner, yielding significant increases in Fpg-sensitive sites above background after incubation for 24 or 48 h with 500 and 250 microM respectively. At both time points the frequency of DNA base modifications exceeded the number of DNA strand breaks. In contrast, neither DNA strand breaks nor Fpg-sensitive sites were detected after treatment with Fe-citrate at concentrations up to 2 microM for 24 or 48 h; this inactivity of Fe-citrate was independent of the molar ratio of iron to ligand (1:1, 1:2, 1:10 or 1:20). The results indicate that the cellular damage induced by ferric iron depends strongly on the actual complex applied, possibly due to differences in the intracellular distribution, which in turn may affect the availability of iron for redox reactions at or in close proximity to the DNA.     

Deficient repair of UV-induced DNA damage in immature human T cells

As compared with quiescent lymphocytes, human thymocytes were found in the nucleoid sedimentation assay to be deficient in UV-induced DNA repair. Therefore, the age-related repair deficiency of human lymphocytes can result from the increased proportion of immature T cells occurring with age.     

The amyloid beta protein induces oxidative damage of mitochondrial DNA

A 55-year-old man was admitted to our hospital with of hemoptysis, progression of anemia and renal failure in February, 1996. Idiopathic interstitial pneumonia had been diagnosed and he had been followed at a regional hospital since 1988. On the third day after admission, he suffered from sudden and massive hemoptysis. Goodpasture's syndrome was diagnosed because anti-GBM antibody was detected in serum. A high titer of MPO-ANCA was also recognized simultaneously. Steroid pulse therapy, immunosuppressive therapy, and plasmapheresis were begun, but he died on the 28th hospital day because of severe hypoxemia and multi-organ failure. Histological examination after autopsy revealed crescentic glomerulonephritis with linear deposition of IgG in the glomerular capillary wall, and interstitial pneumonia accompanied by massive alveolar hemorrhage. It was suggested that in this patient, not only anti-GBM antibody but also circulating MPO-ANCA might have participated in the progression of the crescentic glomerulonephritis and alveolar hemorrhage observed in Goodpasture's syndrome.     

Novel DNA binding motifs in the DNA repair enzyme endonuclease III crystal structure

The 1.85 A crystal structure of endonuclease III, combined with mutational analysis, suggests the structural basis for the DNA binding and catalytic activity of the enzyme. Helix-hairpin-helix (HhH) and [4Fe-4S] cluster loop (FCL) motifs, which we have named for their secondary structure, bracket the cleft separating the two alpha-helical domains of the enzyme. These two novel DNA binding motifs and the solvent-filled pocket in the cleft between them all lie within a positively charged and sequence-conserved surface region. Lys120 and Asp138, both shown by mutagenesis to be catalytically important, lie at the mouth of this pocket, suggesting that this pocket is part of the active site. The positions of the HhH motif and protruding FCL motif, which contains the DNA binding residue Lys191, can accommodate B-form DNA, with a flipped-out base bound within the active site pocket. The identification of HhH and FCL sequence patterns in other DNA binding proteins suggests that these motifs may be a recurrent structural theme for DNA binding proteins.     

Long-range and short-range oxidative damage to DNA: photoinduced damage to guanines in ethidium-DNA assemblies

Short-range and long-range photoreactions between ethidium and DNA have been characterized. While no DNA reaction is observed upon excitation into the visible absorption band of ethidium, higher-energy irradiation (313-340 nm) leads both to direct strand cleavage at the 5'-G of 5'-GG-3' doublets and to piperidine-sensitive lesions at guanine. This reactivity is not consistent with oxidation of guanine by either electron transfer or singlet oxygen as shown by comparison with reactions of a rhodium intercalator and methylene blue, respectively. By covalently tethering ethidium to one end of a DNA duplex, we demonstrate the presence of two distinct reactions, one short-range and the other long-range. The short-range reaction involves a covalent modification of guanine by ethidium, based upon HPLC analysis of the nucleoside products and studies with ethidium derivatives. The long-range reaction is entirely consistent with oxidation of guanine by DNA-mediated electron transfer. The yield of this electron-transfer reaction is not attenuated with distance; equal yields of guanine damage are observed at a proximal (17 A Et-GG separation) and distal (44 A Et-GG separation) site. These results are quite similar to those previously observed with a covalently tethered rhodium photooxidant and underscore the unique ability of the DNA base stack to facilitate long-range electron transfer so as to effect oxidative damage from a distance.     

In vivo dual effects of vitamin C on paraquat-induced lung damage: dependence on released metals from the damaged tissue

Vitamin C, a potent antioxidant, can act as a pro-oxidant in the presence of free transition metal ions by accelerating the Fenton reaction. An in vivo pro-oxidant role of vitamin C has been suggested, but direct evidence for it is scant. Here, we report the dual role of vitamin C on paraquat-induced lung injury, which appears to depend on the metal ions released from damaged cells. Vitamin C (10 mg/kg) given at the time when the extensive tissue damage was in progress aggravated the oxidative damage, while it protected against the damage when given before the initiation of the damage. The extent of oxidative tissue damage was monitored by measuring the expiratory ethane, one of the hydrocarbons produced during lipid peroxidation. Deferoxamine, given intraperitoneally as a bolus dose of 50 mg/kg, completely blocked the aggravation of oxidative damage by vitamin C. Moreover, deferoxamine unmasked the antioxidant effect of vitamin C. The results show that vitamin C can either aggravate or alleviate the oxidative tissue damage depending on the presence of metal ions released from damaged cells.