Influence of cellular differentiation on repair of ultraviolet-induced DNA damage in murine proadipocytes

The effects of cellular differentiation on the repair of DNA damage induced by uv radiation were investigated in the murine 3T3-T proadipocyte cell culture system. Upon exposure to human plasma, actively cycling 3T3-T cells (stem cells) undergo growth arrest, which is followed by terminal differentiation into lipid-laden adipocytes. In response to uv irradiation, the level of unscheduled DNA synthesis is significantly lower in adipocytes as compared to stem cells. The alkaline elution assay was used to monitor the appearance of repair-induced strand breaks in 3T3-T cells after uv irradiation. DNA strand breaks were detected in stem cells by 4 min post-uv with essentially no further increase after 8 min. When terminally differentiated adipocytes were irradiated and allowed to repair, however, more strand breaks were present at 4 min and, in marked contrast to stem cells, continued to accumulate in adipocytes for at least 16 min post-uv. Inhibition of repair-replication with hydroxyurea and cytosine arabinoside significantly increased accumulation of repair-induced strand breaks in stem cells, yet had little effect on this accumulation in adipocytes. For stem cells and adipocytes, incision activity was linear out to at least 10 Jm-2 without saturation. These data suggested that 3T3-T cell differentiation is accompanied by a defect in some postincision process of the excision-repair pathway.     

DNA repair efficiency for ultraviolet induced damage

Ultraviolet radiation causes lesions in bacterial DNA which are repaired by several enzyme systems. Wide variations in the efficiency of repair for differentE. Coli strains are inadequately explained by a simple presence or absence of one or more repair systems. It is proposed that a major factor in the variations is the sensitivity of the repair systems themselves to ultraviolet induced interactions between proteins and the repair enzyme cistrons. An analytic approach is applied to pre-existing data to establish the numbers of thymine and cytosine bases in the repair cistrons, lending support to the model. The findings imply that bacteria will become sensitive to UV upon inhibition of one of four amino acids.     

Immunoanalysis of ultraviolet radiation induced DNA damage and repair within specific gene segments of plasmid DNA.

The region-specific heterogeneity of repairing DNA damage has been established in several biological systems. A flexible and sensitive approach, based upon DNA damage specific antibodies, is described to monitor the repair of specific lesions within discrete genomic segments. Membrane transblotted DNA restriction fragments are immunoanalyzed for the initial formation and repair of 254 nm radiation induced pyrimidine dimers. Sensitivity of dimer immunodetection increases proportional to fragment concentration and size. Antibody binding was detectable in a 0.5 kb fragment obtained from approx. 100 ng of restriction digested phage lambda DNA irradiated with 50 J m-2. Dimers within larger fragments (greater than 5 kb) could be detected at ultraviolet doses as low as 1 to 2 J m-2. To determine the occurrence of preferential repair in prokaryotic cells, damage was assessed in DNA sequences established in various Escherichia coli strains. In vivo repair of 8.9 kb vector and 6.4 and 3.2 kb gene inserts occurred with an approximate t1/2 of 45 min in UvrABC excision repair-proficient strains. Antibody binding sites were retained by DNA within repair-deficient strains. Compared to UvrABC, the repair of DNA fragments mediated by T4 endonuclease V was rapid and complete within 30 min of cellular irradiation. The efficient repair in DenV+ strain is attributable to a highly processive repair enzyme rather than to selective repair of actively replicating target genes. The results demonstrate the exceptional ability of antibodies specific for altered biomolecular lesions to map damage and repair in gene segments episomally established within cells.     

Pesticide induced DNA damage and its repair in cultured human cells.

The effects of pesticides on the induction of unscheduled DNA synthesis in SV-40 transformed human cells (VA-4) in culture with and without metabolic activation by liver microsomes was studied. Results showed that ten of the thirteen compounds examined either directly or upon metabolic activation induced unscheduled DNA synthesis in the human cell system used. The DNA repair kinetics and size of the repaired regions resulting from treatment with four of the chemicals (Carbaryl, Chlordane, Dieldrin and 2.4-D Fluid) were studied by 313 nm photolysis of repaired regions containing bromodeoxyuridine (BUdR). The size of the repaired regions differed between compounds but could generally be classified as either of the X-ray (short) or UV-type (long).     

Influence of iron-overload on DNA damage and its repair in human leukocytes in vitro

Iron is an important element that modulates the production of reactive oxygen species, which are thought to play a causative role in biological processes such as mutagenesis and carcinogenesis. The potential genotoxicity of dietary iron has been seldom studied in human leukocyte and only few reports have investigated in human colon tumor cells. Therefore, DNA damage and repair capacity of human leukocytes were examined using comet assay for screening the potential toxicity of various iron-overloads such as ferric-nitrilotriacetate (Fe-NTA), FeSO(4), hemoglobin and myoglobin, and compared with 200μM of H(2)O(2) and HNE. The iron-overloads tested were not cytotoxic in the range of 10-1000 microM by trypan blue exclusion assay. The exposure of leukocytes to Fe-NTA (500 and 1000 microM), FeSO(4) (250-1000 microM), hemoglobin (10 microM) and myoglobin (250 microM) for 30 min induced significantly higher DNA damage than NC. Treatment with 500 and 1000 microM of Fe-NTA showed a similar genotoxic effect to H(2)O(2), and a significant higher genotoxic effect than HNE. The genotoxicity of FeSO(4) (250-1000 microM), hemoglobin (10 microM) and myoglobin (250 microM) was not significantly different from that of H(2)O(2) and HNE. Iron-overloads generated DNA strand break were rejoined from the first 1h. Their genotoxic effect was not observed at 24h. These data from this study provide additional information on the genotoxicity of iron-overloads and self-repair capacity in human leukocytes.     

Influence of polymorphisms in DNA repair genes XPD, XRCC1 and MGMT on DNA damage induced by gamma radiation and its repair in lymphocytes in vitro

DNA single-strand breaks (SSBs) were quantified by single-cell gel electrophoresis and micronucleated and apoptotic cells were quantified by microscopic assays in peripheral blood lymphocytes after irradiation on ice with 2 Gy of 60Co gamma radiation, and their association with polymorphisms of genes that encode proteins of different DNA repair pathways and influence cancer risk (XPD codon 312Asp --> Asn and 751Lys --> Gln, XRCC1 399Arg --> Gln, and MGMT 84Leu --> Phe) was studied. In unirradiated lymphocytes, SSBs were significantly more frequent in individuals older than the median age (52 years) (P = 0.015; n = 81), and the frequency of apoptotic or micronucleated cells was higher in individuals with alleles coding for Asn at XPD 312 or Gln at 751 (P = 0.030 or 0.023 ANOVA, respectively; n = 54). The only polymorphism associated with the background SSB level was MGMT 84Phe (P = 0.04, ANOVA; n = 66). After irradiation, SSB levels and repair parameters did not differ significantly with age or smoking habit. The SSB level varied more than twofold and the repair rate and level of unrepaired SSBs more than 10-fold between individuals. The presence of variant alleles coding for Asn at XPD 312 was associated with more radiation-induced SSBs (P = 0.014) and fewer unrepaired SSBs (P = 0.008), and the phenotype (> median induced SSBs/< median unrepaired SSBs) was seen in the majority of XPD 312Asn/Asn homozygotes; the odds ratio for variant homozygotes to show this phenotype was 5.2 (95% confidence interval 1.4-19.9). The hypothesis is discussed that XPD could participate in repair of ionizing radiation-induced DNA damage. While it cannot be excluded that the effects observed are due to cosegregating polymorphisms or that the responses of lymphocytes are not typical of other cell types, the results suggest that polymorphism of DNA repair genes, particularly XPD, is one factor implicated in the variability of responses to ionizing radiation between different individuals.     

Radiation-induced DNA damage and its repair

Application of modern methods of organic chemistry and recombinant DNA technologies has provided new insights in the field of DNA radiation damage and its repair. An overview of the chemical nature of the lesions inflicted on DNA by ionizing radiation is presented. The structures of 29 different DNA modified base or sugar residues are shown in comprehensive formation schemes. A fraction of radiation-induced modified bases is spontaneously released from the DNA chain during irradiation. Another part remains attached to the DNA chain backbone and for its characterization mild formic acid or enzymatic hydrolysis have been used. Starting from the chemical formulae of the altered base residues, the specific repair enzymes and their modes of action are discussed. Various glycosylases and endonucleases have been purified to homogeneity, and in some cases the gene which encodes the protein cloned. Using methods derived from Maxam and Gilbert sequencing procedures and DNA fragment 32P-labelled at one end, it has been shown that the alkali-labile sites in DNA induced by radiation are strongly dependent on the DNA base sequence. Enzymatic methods have been used to analyse the DNA base defects produced by gamma-irradiation of cells under in vivo conditions. Structures of modified bases were the same as those observed when DNA was irradiated in aqueous solution.     

Decreased repair of radiation-induced DNA double-strand breaks with cellular differentiation.

Although the majority of mammalian cells in situ are terminally differentiated, most DNA repair studies have used proliferating cells. In an attempt to understand better the relationship between differentiation and DNA repair, we have used the murine 3T3-T proadipocyte cell line. In this model system, proliferating (stem) cells undergo growth arrest (GD cells) and subsequently terminally differentiate into adipocytes when exposed to media containing platelet-depleted human plasma. Pulsed-field gel electrophoresis was used to evaluate the induction and repair of DNA double-strand breaks (DSBs) after ionizing radiation. The levels of radiation-induced DSBs in GD and terminally differentiated cells were similar, but in both cases greater than those found in stem cells at each radiation dose tested (0 to 40 Gy); these differences appear to be due to growth arrest in G1 phase. DNA DSBs were repaired with biphasic kinetics for each cell type. For terminally differentiated cells 25% of DNA DSBs remained unrejoined compared with < 10% for GD and stem cells after a repair time of 4 h. These data indicate that terminal differentiation of 3T3-T cells is associated with a reduction in the repair of ionizing radiation-induced DNA DSBs.     

Analysis of DNA damage and repair accompanying differentiation in the intestinal crypt.

The ability to process damaged DNA may vary between cells depending on their differentiated status. However, there is little in vivo data available and it is not intuitively obvious how the activity of specific repair pathways may vary between different subpopulations (e.g. stem cells and proliferative, committed and differentiated cells) of a particular tissue. To obtain such information for the intestinal epithelium, we have developed an assay that detects differences in the way different regions of the crypt (stem, proliferative and maturation zones) respond to DNA damage. The assay is a variant of the ''comet'' assay, which detects DNA strand breaks by measuring the proportion of DNA migrating from individual cells, or in this case intact isolated crypts, in an electrophoretic field. The method is quantitative, with the amount of migrating DNA being proportional to the number of strand breaks. Isolated crypts are repair competent and spatial differences are apparent with some agents. The assay has the potential to characterize the repair properties of cells at different stages of differentiation within the crypt, determine the characteristics that might predispose them to damage and may help in understanding the route of stem cell mutation.     

Replicative bypass repair of ultraviolet damage to DNA of mammalian cells: Caffeine sensitive and caffeine resistant mechanisms

Replicative bypass repair of UV damage to DNA was studied in wide variety of human, mouse and hamster cells in culture. Survival curve analysis revealed that in established cell lines (mouse L, Chinese hamster V79, HeLa S3 and SV40-transformed xeroderma pigmentosum (XP)), post-UV caffeine treatment potentiated cell killing by reducing the extrapolation number and mean lethal UV fluence (Do). In the Do reduction as the result of random inactivation by caffeine of sensitive repair there were marked clonal differences among such cell lines, V79 being most sensitive to caffeine potentiation. However, other diploid cell lines (normal human, excision-defective XP and Syrian hamster) exhibited no obvious reduction in Do by caffeine. In parallel, alkaline sucrose sedimentation results showed that the conversion of initially smaller segments of DNA synthetized after irradiation with 10 J/m² to high-molecular-weight DNA was inhibited by caffeine in transformed XP cells, but not in the diploid human cell lines. Exceptionall, diploid XP variants had a retarded ability of bypass repair which was drastically prevented by caffeine, so that caffeine enhanced the lethal effect of UV. Neutral CsCl study on the bypass repair mechanism by use of bromodeoxyuridine for DNA synthesis on damaged template suggests that the pyrimidine dimer acts as a block to replication and subsequently it is circumvented presumably by a new process involving replicative bypassing following strand displacement, rather than by gap-filling de novo. This mechanism worked similarly in normal and XP cells, whether or not caffeine was present, indicating that excision of dimer is not always necessary. However, replicative became defective in XP variant and transformed XP cells when caffeine was present. It appears, therefore, that the replicative bypass repair process is either caffeine resistant or sensitive, depending on the cell type used, but not necessarily on the excision repair capability.     

Restricted repair of aflatoxin B1 induced damage in alpha DNA of monkey cells

We have investigated the processing of adducts formed by covalent binding of aflatoxin B1 (AFB1) to DNA in confluent cultures of African green monkey cells. Repair synthesis elicited by AFB1 adducts was deficient in alpha DNA sequences compared to that in bulk DNA, although the initial levels of modification were the same for these DNAs. The removal of the primary initial adduct, AFB1-N7-Guanine, was deficient in alpha DNA and the kinetics of its loss resembled those previously reported for removal from total DNA in xeroderma pigmentosum cells of complementation group A. Spontaneous loss of the AFB1 moiety or the concomitant loss of the guanine to yield an apurinic site account for these results. The formation of the more chemically stable secondary product, AFB1-triamino-Pyrimidine, occurred more rapidly and to a greater extent in alpha DNA than in bulk DNA, probably because of slower removal of the primary product. The excision repair patch size for AFB1 adducts in alpha DNA was only 10 nucleotides compared to 20 nucleotides for repair of AFB1 adducts in bulk DNA. Irradiation of cells with low doses of UV prior to or immediately after treatment with AFB1 increased the rate and extent of removal of AFB1 adducts from alpha DNA to the levels found in the bulk DNA, indicating that the formation of pyrimidine dimers or their repair may alter the chromatin structure of alpha DNA sufficiently to facilitate its repair.     

Crystal structure of the DNA repair enzyme ultraviolet damage endonuclease

The ultraviolet damage endonuclease (UVDE) performs the initial step in an alternative excision repair pathway of UV-induced DNA damage, nicking immediately adjacent to the 5' phosphate of the damaged nucleotides. Unique for a single-protein DNA repair endonuclease, it can detect different types of damage. Here we show that Thermus thermophilus UVDE shares some essential structural features with Endo IV, an enzyme from the base excision repair pathway that exclusively nicks at abasic sites. A comparison between the structures indicates how DNA is bound by UVDE, how UVDE may recognize damage, and which of its residues are involved in catalysis. Furthermore, the comparison suggests an elegant explanation of UVDE's potential to recognize different types of damage. Incision assays including point mutants of UVDE confirmed the relevance of these conclusions.     

Transient conformation changes in chromatin during excision repair of ultraviolet damage to DNA.

DNA labeled for 15 minutes during UV induced repair synthesis is two-fold more sensitive to micrococcal nuclease than the bulk nuclear DNA. As the length of the labeling period increases from 15 minutes to 4 hours the nuclease sensitivity of repair labeled DNA approaches that of bulk chromatin. Pulse-chase experiments indicate that the nuclease sensitivity of the repaired DNA labeled during a brief pulse decreases with a half-life of about 15 minutes. In contrast to previous interpretations, we consider these results to mean that immediately after synthesis, chromatin labeled during repair has a conformation which renders it more susceptible to nuclease digestion than the bulk chromatin. With time these repaired regions are assembled into a nucleosome structure with normal nuclease sensitivity.     

Sequence-specific DNA damage induced by ultraviolet A-irradiated folic acid via its photolysis product

DNA damage mediated by photosensitizers participates in solar carcinogenesis. Fluorescence measurement and high-performance liquid chromatography analysis demonstrated that photoirradiated folic acid, one of the photosensitizers in cells, generates pterine-6-carboxylic acid (PCA). Experiments using 32P-labeled DNA fragments obtained from a human gene showed that ultraviolet A-irradiated folic acid or PCA caused DNA cleavage specifically at consecutive G residues in double-stranded DNA after Escherichia coli formamidopyrimidine-DNA glycosylase or piperidine treatment. The amount of 8-oxo-7,8-dihydro-2(')-deoxyguanosine formed through this DNA photoreaction in double-stranded DNA exceeded that in single-stranded DNA. Kinetic studies suggested that DNA damage is caused mainly by photoexcited PCA generated from folic acid rather than by folic acid itself. In conclusion, photoirradiated folic acid generates PCA, which induces DNA photooxidation specifically at consecutive G residues through electron transfer. Excess intake of folic acid supplements may increase a risk of skin cancer by solar ultraviolet light.     

The reactivity of the 2-deoxyribonolactone lesion in single-stranded DNA and its implication in reaction mechanisms of DNA damage and repair.

The formal C1'-oxidation product, 2-deoxyribonolactone, is formed as a result of DNA damage induced via a variety of agents, including gamma-radiolysis and the enediyne antitumor antibiotics. This alkaline labile lesion may also be an intermediate during DNA damage induced by copper-phenanthroline. Oligo-nucleotides containing this lesion at a defined site were formed via aerobic photolysis of oligonucleotides containing a photolabile ketone, and were characterized by gel electrophoresis and electrospray mass spectrometry (ESI-MS). Treatment of oligo-nucleotides containing the lesion with secondary amines produces strand breaks consisting of 3'-phosphate termini, and products which migrate more slowly in polyacrylamide gels. MALDI-TOF mass spectrometry analysis indicates that the slower moving products are formal adducts of the beta-elimination product resulting from 2-deoxyribonolactone and one molecule of amine. The addition of beta-mercapto-ethanol to the reaction mixture produces thiol adducts as well. The stability of these adducts suggests that they cannot be the labile species characterized by gel electrophoresis in copper-phenanthroline-mediated strand scission. The characterization of these adducts by mass spectrometry also provides, by analogy, affirmation of proposals regarding the reactivity of nucleophiles with the beta-elimination product of abasic sites. Finally, the effects of this lesion and the various adducts on DNA repair enzymes are unknown, but their facile generation from oligonucleotides containing a photolabile ketone suggests that such issues could be addressed.     

Measurement of cellular repair activities for oxidative DNA damage

Two related assays capable of determining cell extract repair activities for different oxidative lesions in DNA are described. Both assays measure the incorporation of radiolabeled nucleotides during repair of an oxidatively damaged template in a cell-free system. The assays differ in the type of oxidative damage present in the DNA. In one, singlet oxygen is used to generate predominantly 8-oxo-2'-deoxyguanosine lesions. In the other, hydroxyl radicals are used to generate a broad spectrum of damage including oxidized bases and strand breaks. Assay conditions were adjusted to ensure that radiolabel incorporation was directly proportional to cell extract repair activity. These assays represent sensitive tools for investigating the regulation of repair systems for oxidative DNA damage.     

Molecular mechanisms of DNA damage and repair: progress in plants

Despite stable genomes of all living organisms, they are subject to damage by chemical and physical agents in the environment (e.g., UV and ionizing. radiations, chemical mutagens, fungal and bacterial toxins, etc.) and by free radicals or alkylating agents endogenously generated in metabolism. DNA is also damaged because of errors during its replication. The DNA lesions produced by these damaging agents could be altered base, missing base, mismatch base, deletion or insertion, linked pyrimidines, strand breaks, intra- and inter-strand cross-links. These DNA lesions could be genotoxic or cytotoxic to the cell. Plants are most affected by the UV-B radiation of sunlight, which penetrates and damages their genome by inducing oxidative damage (pyrimidine hydrates) and cross-links (both DNA protein and DNA-DNA) that are responsible for retarding the growth and development. The DNA lesions can be removed by repair, replaced by recombination, or retained, leading to genome instability or mutations or carcinogenesis or cell death. Mostly organisms respond to genome damage by activating a DNA damage response pathway that regulates cell-cycle arrest, apoptosis, and DNA repair pathways. To prevent the harmful effect of DNA damage and maintain the genome integrity, all organisms have developed various strategies to either reverse, excise, or tolerate the persistence of DNA damage products by generating a network of DNA repair mechanisms. A variety of different DNA repair pathways have been reported that include direct reversal, base excision repair, nucleotide excision repair, photoreactivation, bypass, double-strand break repair pathway, and mismatch repair pathway. The direct reversal and photoreactivation require single protein, all the rest of the repair mechanisms utilize multiple proteins to remove or repair the lesions. The base excision repair pathway eliminates single damaged base, while nucleotide excision repair excises a patch of 25- to 32-nucleotide-long oligomer, including the damage. The double-strand break repair utilizes either homologous recombination or nonhomologous endjoining. In plant the latter pathway is more error prone than in other eukaryotes, which could be an important driving force in plant genome evolution. The Arabidopsis genome data indicated that the DNA repair is highly conserved between plants and mammals than within the animal kingdom, perhaps reflecting common factors such as DNA methylation. This review describes all the possible mechanisms of DNA damage and repair in general and an up to date progress in plants. In addition, various types of DNA damage products, free radical production, lipid peroxidation, role of ozone, dessication damage of plant seed, DNA integrity in pollen, and the role of DNA helicases in damage and repair and the repair genes in Arabidopsis genome are also covered in this review.