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PubMed2005

ATM signaling and genomic stability in response to DNA damage.

DNA double strand breaks represent the most threatening lesion to the integrity of the genome in cells exposed to ionizing radiation and radiomimetic chemicals. Those breaks are recognized, signaled to cell cycle checkpoints and repaired by protein complexes. The product of the gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) plays a central role in the recognition and signaling of DNA damage. ATM is one of an ever growing number of proteins which when mutated compromise the stability of the genome and predispose to tumour development. Mechanisms for recognising double strand breaks in DNA, maintaining genome stability and minimizing risk of cancer are discussed.

Animals↗

PubMed1999

Induction by adozelesin and hydroxyurea of origin recognition complex-dependent DNA damage and DNA replication checkpoints in Saccharomyces cerevisiae.

DNA damaging agents induce a conserved intra-S-phase checkpoint that inhibits DNA replication in eukaryotic cells. To better understand this checkpoint and its role in determining the efficacy of antitumor drugs that damage DNA, we examined the effects of adozelesin, a DNA-alkylating antitumor agent that has a profound inhibitory effect on initiation of DNA replication in mammals, on the replication of Saccharomyces cerevisiae chromosomes. Adozelesin inhibited initiation of S. cerevisiae DNA replication by inducing an intra-S-phase DNA damage checkpoint. This inhibitory effect was abrogated in orc2-1 cells containing a temperature-sensitive mutation in a component of the origin recognition complex (ORC) that also causes a defect in initiation. The orc2-1 mutation also caused a defect in a checkpoint that regulates the activation of origins in late S phase in cells treated with hydroxyurea. Defects in both initiation and checkpoint regulation in the orc2-1 strain were suppressed by deletion of a gene encoding a putative acetyltransferase, SAS2. Adozelesin also induced a cellular response that requires a function of ORC in G(1). A similar G(1)-specific response in mammals may contribute to the cytotoxic and antitumor properties of this and other DNA-damaging drugs.

Antineoplastic Agents, Alkylating↗

PubMed1999

Modulation of DNA damage and DNA repair in chromatin.

DNA is packaged in the highly compact and dynamic structure of chromatin in eukaryotic cells. It is generally accepted that DNA processing events in the nucleus, such as transcription, replication, recombination, and repair, are restricted by this packaging. For some processes (e.g., transcription), the chromatin fiber is "preset" in a more open structure to allow access of proteins to specific regions of DNA within this structural hierarchy. These regions contain modified nucleosomes that accommodate a less compact state of chromatin and allow access to specific regions of DNA. DNA repair proteins, however, must access DNA lesions in all structural domains of chromatin after sudden insult to the genome. Damaged DNA must be recognized, removed, and replaced by repair enzymes at all levels of chromatin packaging. Therefore, the modulation of DNA damage and its repair in chromatin is crucial to our understanding of the fate of potential mutagenic and carcinogenic lesions in DNA. In this review, we discuss the modulation of DNA damage and DNA repair by chromatin structure, and the modulation of chromatin structure by these events.

Animals↗

PubMed1995

Enhanced p53 activity and accumulation in response to DNA damage upon DNA transfection.

In response to DNA damage the wild-type tumor suppressor protein p53 accumulates in the nucleus of rodent and primate cells. To investigate the minimal requirement for this reaction the cellular DNA was restricted by two alternative ways: (i) by calicheamicin gamma 1, an enediyne, which causes direct, sequence-specific DNA damage, as shown by fluorimetric analysis of DNA unwinding and by poly(ADP-ribose) polymerase activation. The dose-dependent DNA damage correlated with the nuclear p53 accumulation. In addition, restriction was generated (ii) by the intracellular introduction of the restriction enzyme PvuII, which generates blunt-ended DNA breaks, applying a mild hypotonic shock (pellet method). Previous transfection of linear or circular, single- or ds, DNA, followed by mitomycin C-treatment, lead to a dramatic increase in nuclear p53 accumulation and p53 activity according to electrophoretic mobility shift analysis. The nature of transfected DNA was irrelevant for enhanced accumulation. The data suggest, that the cellular p53 response to DNA damage is sensitized by uptake of exogenous DNA.

Aminoglycosides↗

PubMed2004

Overproduction, crystallization and preliminary crystallographic analysis of a novel human DNA-repair enzyme that recognizes oxidative DNA damage.

DNA glycosylases repair oxidative DNA damage caused by free radicals. Recently, NEIL1, a human homolog of Escherichia coli DNA glycosylase endonuclease VIII, has been identified and shown to exhibit broad substrate specificity for a variety of types of pyrimidine-base damage. An active C-terminal deletion construct of NEIL1 was overexpressed in E. coli and crystallized. The unliganded NEIL1 crystallizes in space group R3, with unit-cell parameters a = b = 132.2, c = 51.1 A. Complete data sets were collected from native, selenomethionyl and iodinated NEIL1 to 2.1, 2.3 and 2.4 angstroms, respectively.

Crystallography, X-Ray↗

PubMed2001

UV-damaged DNA-binding protein in the TFTC complex links DNA damage recognition to nucleosome acetylation.

Initiation of transcription of protein-encoding genes by RNA polymerase II (Pol II) was thought to require transcription factor TFIID, a complex comprised of the TATA box-binding protein (TBP) and TBP-associated factors (TAF(II)s). In the presence of TBP-free TAF(II) complex (TFTC), initiation of Pol II transcription can occur in the absence of TFIID. TFTC containing the GCN5 acetyltransferase acetylates histone H3 in a nucleosomal context. We have identified a 130 kDa subunit of TFTC (SAP130) that shares homology with the large subunit of UV-damaged DNA-binding factor. TFTC preferentially binds UV-irradiated DNA, UV-damaged DNA inhibits TFTC-mediated Pol II transcription and TFTC is recruited in parallel with the nucleotide excision repair protein XP-A to UV-damaged DNA. TFTC preferentially acetylates histone H3 in nucleosomes assembled on UV-damaged DNA. In agreement with this, strong histone H3 acetylation occurs in intact cells after UV irradiation. These results suggest that the access of DNA repair machinery to lesions within chromatin may be facilitated by TFTC via covalent modification of chromatin. Thus, our experiments reveal a molecular link between DNA damage recognition and chromatin modification.

Acetylation↗

PubMed1979

DNA damage and DNA repair in cultured human cells exposed to chromate.

DNA damage and DNA repair have been observed in cultured human skin fibroblasts exposed to potassium chromate but not to a chromic glycine complex. DNA repair synthesis (unscheduled incorporation of [3H]thymidine (TdR)) was measured in cells during or following exposure to chromate and was significant for chromate concentrations above 10(-6) M. Maximal DNA repair was observed at about 10(-4) M chromate. DNA repair capacity was found to be saturated at this concentration. Chromate was stable for at least 8 h in culture medium and produced approximately a linear increase in repair with duration of exposure. DNA damage as determined by alkaline sucrose gradient sedimentation was detected after treatment for 1.5 h with 5 . 10(-4) M chromate. Exposure to 10(-7) M chromate solution for 7 days inhibited colony formation while acute (1 h) treatment was toxic at 5 . 10(-6) M. The chromic glycine complex was toxic above 10(-3) M for a 1-week exposure but was not observably toxic after a 1-h treatment. These results indicate that chromate and not chromic compounds may be the carcinogenic form for man. The nature of the ultimate carcinogen is discussed. These findings illustrate the utility of the DNA repair technique to study the effects on human cells of inorganic carcinogens and mutagens.

Adult↗

PubMed1997

DNA-damage & DNA-repair in ageing brain.

The validity of DNA damage and repair hypothesis of ageing has been examined with special reference to the brain. As far as the accumulation of DNA damage is concerned, the data appear to overwhelmingly support the hypothesis. However, the results of attempts to demonstrate a decline in DNA repair capacity as a function of age are conflicting. However, more recent observations are in support of age-dependent deterioration in DNA-repair capacity. Possible reasons for this discrepancy and the usefulness of conducting DNA damage and repair studies in a model system of isolated neuronal cells, are discussed. It is suggested that assessment of repair capacity of brain with respect to a specific damage in a designated gene might yield more definitive answers regarding the role of DNA repair potential in the ageing process and as a longevity assurance system.

Aging↗

PubMed1993

Delayed chromosomal instability induced by DNA damage.

DNA damage induced by ionizing radiation can result in gene mutation, gene amplification, chromosome rearrangements, cellular transformation, and cell death. Although many of these changes may be induced directly by the radiation, there is accumulating evidence for delayed genomic instability following X-ray exposure. We have investigated this phenomenon by studying delayed chromosomal instability in a hamster-human hybrid cell line by means of fluorescence in situ hybridization. We examined populations of metaphase cells several generations after expanding single-cell colonies that had survived 5 or 10 Gy of X rays. Delayed chromosomal instability, manifested as multiple rearrangements of human chromosome 4 in a background of hamster chromosomes, was observed in 29% of colonies surviving 5 Gy and in 62% of colonies surviving 10 Gy. A correlation of delayed chromosomal instability with delayed reproductive cell death, manifested as reduced plating efficiency in surviving clones, suggests a role for chromosome rearrangements in cytotoxicity. There were small differences in chromosome destabilization and plating efficiencies between cells irradiated with 5 or 10 Gy of X rays after a previous exposure to 10 Gy and cells irradiated only once. Cell clones showing delayed chromosomal instability had normal frequencies of sister chromatid exchange formation, indicating that at this cytogenetic endpoint the chromosomal instability was not apparent. The types of chromosomal rearrangements observed suggest that chromosome fusion, followed by bridge breakage and refusion, contributes to the observed delayed chromosomal instability.

Animals↗

PubMed1994

Recognition of platinum-induced DNA damage by nuclear proteins: screening for mechanisms.

The association of high mobility group (HMG) proteins with cisplatin-damaged DNA has attracted considerable interest because of a possible relationship with drug-acquired resistance and the repair of DNA damage caused by this important chemotherapeutic agent. We have further characterized the binding of HMG proteins to cisplatin-damaged DNA using a modification of the damaged DNA affinity precipitation assay (DDAP) and proteins isolated from the nuclei of V79 cells. HMG proteins recognized cisplatin adducts only in double-stranded DNA sequences. Pre-treatment of cells with cisplatin (1 microM) prior to protein extraction decreased the apparent yield of HMG proteins. However, the pre-treatment of isolated protein did not prevent recognition of the DNA adducts. To investigate the possible role of HMG proteins in toxicity and resistance, we have extended the DDAP method to study other platinum agents, some of which are active in cisplatin-resistant cells. A comparison of trans- and cis-[PtCl2(NH3)quinoline] is presented as an example. HMG proteins recognized DNA damage caused by the cis, but not the trans isomer. However, the trans isomer is known to be significantly more toxic and is highly active in cisplatin-resistant cells, suggesting a mechanism of action different from cisplatin. Therefore, the toxicity of trans-[PtCl2(NH3)quinoline] appears to be unrelated to the recognition of damage by HMG proteins. The DDAP assay may provide an additional screen for new mechanisms of cytotoxic platinum agents.

Animals↗

PubMed1989

Urinary 8-hydroxy-2'-deoxyguanosine as a biological marker of in vivo oxidative DNA damage.

DNA is subject to constant oxidative damage from endogenous oxidants. The oxidized DNA is continuously repaired and the oxidized bases are excreted in the urine. A simple routine analytical procedure is described for urinary 8-hydroxy-2'-deoxyguanosine, an oxidative DNA damage adduct, as an indicator of oxidative damage in humans and rodents. This adduct was purified from human urine and characterized. The described assay employs a series of solid-phase extraction steps that separate 8-hydroxy-2'-deoxyguanosine from other urinary constituents, followed by analysis by gradient reversed-phase HPLC coupled to a dual-electrode high-efficiency electrochemical detection system. Analysis of urine from three species by this method indicates that mice excrete approximately 3.3-fold more 8-hydroxy-2'-deoxyguanosine than humans (582 vs. 178 residues per cell per day), a result that supports the proposal that oxidative damage to DNA increases in proportion to species-specific basal metabolic rates.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2005

DNA damage and DNA adduct formation in rat tissues following oral administration of acrylamide.

Acrylamide is present as a contaminant in the human diet in heated food products. It has been found to be carcinogenic in laboratory rats and has been classified as probably carcinogenic in humans. In order to clarify the possible involvement of a primary genotoxic mechanism in acrylamide-induced carcinogenicity, both the presence of DNA damage, measured by the comet assay, and the formation of N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GA-Gua) and N3-(2-carbamoyl-2-hydroxyethyl)adenine (N3-GA-Ade), derived from reaction of the active metabolite glycidamide (GA) with the DNA, analyzed by LC/MS/MS, were assessed in selected rat tissues. Rats were administered with single oral doses of acrylamide (18, 36 or 54 mg/kg body weight (b.w.) and the organs (blood leukocytes, brain, bone marrow, liver, testes and adrenals) were sampled at different times after treatment. Results from GA-induced DNA adduct measurements indicated a relatively even organ distribution of the adducts in brain, testes and liver. Organ-specificity in acrylamide carcinogenesis can therefore not be explained by a selective accumulation of GA-DNA adducts in the target organs, at least not after a single dose exposure. The DNA adduct profiles and half-lives were similar in the different organs; except that the N3-GA-Ade adduct was more rapidly removed from tissues than the N7-GA-Gua adduct. Increased extent of DNA migration, as measured by the in vivo rat comet assay, was found in brain and testes, and these specific results seem to be in accordance with the known organ-specificity in acrylamide carcinogenesis in rat. Only weak and transient DNA damage was recorded in the liver, bone marrow and adrenals. The DNA-damaging effect of the compound observed in the blood leukocytes could be a simple biomarker of acrylamide exposure and genotoxicity.

Acrylamide↗

PubMed1999

DNA damage and DNA damage-inducible protein Gadd45 following ischemia in the P7 neonatal rat.

Cerebral ischemia in adult rodents leads to the production of several types of lesions in the genomic DNA, followed by the activation of the damage-response indicator Gadd45. Our purpose was to investigate the structural changes that occur in chromatin DNA and repair processes after ischemic injury in neonatal brain. Neonatal ischemia was induced by the permanent left MCA occlusion in association with 1 h occlusion of the left common carotid artery in 7-day-old Wistar pups. Oligonucleosome fragments that are recognized as the characteristic DNA ladder was observed in a delayed fashion. Double-strand breaks result in high molecular weight fragments of 50- and 300-kbp as demonstrated by pulsed-field gel electrophoresis, and visualized by the TUNEL assay at 24 h of recovery. In contrast, DNA single-strand breaks, shown by the use of DNA polymerase I-mediated biotin-dATP nick translation were not so abundant. Gadd45 immunoreactivity was sequentially increased in vulnerable neurons in the infarct (4 to 24 h) and in sublethally injured neurons in the penumbra (24-48 h). Taken together, these findings suggest that Gadd45 responds to DNA damage following neonatal ischemia. Furthermore, repairing processes seem to be more active in the penumbra and therefore Gadd45 could have also a protective role in cerebral ischemia.

Animals↗

PubMed2000

DNA damage formation, DNA repair, and survival after exposure of DNA repair-proficient and nucleotide excision repair-deficient human lymphoblasts to UVA1 and UVB.

PURPOSE: The comet assay has been used to visualize DNA damage in single cells after exposure to UV light. These comets are commonly thought to reflect transient, repair-induced DNA breaks. The goal of the work presented here was to further characterize the nature of UV-induced comets and to further elucidate DNA damage formation by different wavelengths of ultraviolet light. MATERIALS AND METHODS: Detailed dose-response and time-course experiments with comet formation were carried out with normal and nucleotide excision repair (NER)-deficient xeroderma pigmentosum (XP) lymphoblasts. Irradiation was carried out with low, intermediate, or high doses of UVA1 or UVB, comet formation was observed, cell survival and viability were determined, and UV-induced apoptosis was measured. RESULTS: All responses were dose-dependent. With the intermediate dose of UVA1, a pronounced comet formation was observed without subsequent growth inhibition. Raising levels of porphyrins, which act as photosensitizers, by preincubation with 5-amino-levulinic acid increased comet formation with UVA1, but not with UVB. UVA1-sensitivity and comet formation in XP cells was not significantly different from the normal cells. With UVB no comet formation was seen without subsequent apoptotic cell death. XP cells exhibited the known UVB-hypersensitivity, but their comet formation was not significantly different from that of normal cells. CONCLUSIONS: The findings are compatible with the hypothesis that UV-induced comets represent transient repair-induced DNA breaks. Both, the NER of dimers and the base excision repair of oxidative DNA modifications are thought to contribute to comet formation.

Apoptosis↗

PubMed2000

Oxidative DNA damage precedes DNA fragmentation after experimental stroke in rat brain.

Experimental stroke using a focal cerebral ischemia and reperfusion (FCIR) model was induced in male Long-Evans rats by a bilateral occlusion of both common carotid arteries and the right middle cerebral artery for 30-90 min, followed by various periods of reperfusion. Oxidative DNA lesions in the ipsilateral cortex were demonstrated using Escherichia coli formamidopyrimidine DNA N-glycosylase (Fpg protein)-sensitive sites (FPGSS), as labeled in situ using digoxigenin-dUTP and detected using antibodies against digoxigenin. Because Fpg protein removes 8-hydroxy-2'-deoxyguanine (oh8dG) and other lesions in DNA, FPGSS measure oxidative DNA damage. The number of FPGSS-positive cells in the cortex from the sham-operated control group was 3 +/- 3 (mean +/- SD per mm(2)). In animals that received 90 min occlusion and 15 min of reperfusion (FCIR 90/15), FPGSS-positive cells were significantly increased by 200-fold. Oxidative DNA damage was confirmed by using monoclonal antibodies against 8-hydroxy-guanosine (oh8G) and oh8dG. A pretreatment of RNase A (100 microg/ml) to the tissue reduced, but did not abolish, the oh8dG signal. The number of animals with positive FPGSS or oh8dG was significantly (P<0.01) higher in the FCIR group than in the sham-operated control group. We detected few FPGSS of oh8dG-positive cells in the animals treated with FCIR of 90/60. No terminal UTP nicked-end labeling (TUNEL)-positive cells, as a detection of cell death, were detected at this early reperfusion time. Our data suggest that early oxidative DNA lesions elicited by experimental stroke could be repaired. Therefore, the oxidative DNA lesions observed in the nuclear and mitochondrial DNA of the brain are different from the DNA fragmentation detected using TUNEL.

Animals↗

PubMed1998

(6-4) photolyase: light-dependent repair of DNA damage.

DNA photolyase represents a phenomenal class of DNA repair enzymes in that it harvests the light energy to repair DNA lesions caused by ultraviolet light. Mother Nature evolves two types of photolyases, one specific for repairing cyclobutane pyrimidine dimers and the other for pyrimidine-(6-4)-pyrimidone photoproducts. Together, these two kinds of DNA photolesions account for the majority of ultraviolet light-induced DNA lesions. So far, the basic chemical steps of the enzyme mechanism of the two classes of photolyases appear to be very similar. Therefore, it will be very interesting to uncover the determinants of the different substrate specificity between the two photolyases. In this review, we focus on the discussion of the photolyase specific for repairing pyrimidine-(6-4)-pyrimidone photoproducts mainly because the research of the cyclobutane pyrimidine dimer photolyase has recently been reviewed quite extensively.

Animals↗

PubMed1999

Brain derived neurotrophic factor protects human neuroblastoma cells from DNA damaging agents.

Neurotrophins are required for survival of neurons during development and may act as survival factors to cells undergoing stress. We tested whether brain derived neurotrophic factor (BDNF) protects neuroblastoma (NB) cells from cytotoxic agents using a model NB cell line, NB 1643, which expresses functional tropomyosin related kinase B (TRKB) following treatment with all-trans-retinoic acid. TRKB is the receptor for BDNF. BDNF increases the EC50 values in survival assays for cisplatin, doxorubicin, and topotecan by two to three fold. Thus, BDNF does indeed protect cells drugs that damage DNA. Cisplatin and doxorubicin are used to treat NB. Topotecan is in clinical studies for the treatment of NB. Since these drugs induce DNA damage, we also investigated whether BDNF might afford protection from gamma irradiation. BDNF also induces more than a two fold resistance to gamma irradiation. Since BDNF protects cells from agents with different mechanisms of damaging DNA and resistance, it seems likely that BDNF may alter a common signaling pathway required for cell death initiation by DNA damaging agents.

Antineoplastic Agents↗

PubMed2004

Thermodynamic characterization of the interaction of mutant UvrB protein with damaged DNA.

During the DNA damage recognition of nucleotide excision repair in Escherichia coli the interaction of UvrB protein with damaged DNA ensures the recognition of differences in the intrinsic chemical structures of a variety of adduct molecules in DNA double helix. Our earlier study indicated that a single tyrosine-to-tryptophan mutation at residue 95 converted the UvrB to a protein [UvrB(Y95W)] that is able to bind to a structure-specific bubble DNA substrate, even in the absence of UvrA. Fluorescence spectroscopy therefore was adopted to investigate the biochemical properties and thermodynamics of DNA damage recognition by the mutant protein. We examined the binding of the UvrB(Y95W) mutant protein to a structure-specific 30 bp DNA substrate containing a single fluorescein which serves as both an adduct and a fluorophore. Binding of the protein to the substrate results in a significant reduction in fluorescence. By monitoring the fluorescence changes, binding isotherms were generated from a series of titration experiments at various physiological temperatures, and dissociation constants were determined. Analysis of our data indicate that interaction of UvrB(Y95W) protein with the adduct incurred a large negative change in heat capacity DeltaC(p)(o)(obs) (-1.1 kcal mol(-1) K(-1)), while the DeltaG(o)(obs) was relatively unchanged with temperature. Further study of the binding at various concentrations of KCl showed that on average only about 1.5 ion pairs were involved in formation of the UvrB-DNA complex. Together, these results suggested that hydrophobic interactions are the main driving forces for the recognition of DNA damage by UvrB protein.

Acrylamide↗