Participation of the intracellular enzymes in the control of the mutation process

Summary The influence of repair and replication on the frequency of spontaneous chromosome aberrations and of those induced by gamma-irradiation is reported.Using the technique of labelling DNA with radioactive ³H-thymidine and measuring the radioactivity of DNA isolated from embryos, the time of initiation and the duration of DNA synthesis in barley seeds was studied after the soaking of the seeds had begun. The average duration of each phase of the first DNA synthesis cycle in soaking barley seeds was found to be as follows: pre-DNA synthesis stage, 10–11 hrs; DNA synthesis stage, 8 hrs. After gamma-irradiation, the intensity of DNA synthesis decreased and the beginning of DNA synthesis was delayed.It was found that the inhibition of repair by caffeine led to an increase in the frequency of both spontaneous and induced chromosome aberrations. Caffeine enhanced several times the frequency of chromosome and chromatid aberrations at the time of the maximal activity of repair enzymes. During DNA replication, caffeine had a lower effect on the realization of premutational lesions.An inhibitor of DNA replication — hydroxyurea — had no influence on the frequency of spontaneous chromosome aberrations during the replication period, whereas after gamma-irradiation, hydroxyurea enhanced the frequency of aberrations mainly at the stage of DNA replication.The relatively small mutagenic action of both agents (caffeine and hydroxyurea) was observed during all stages of the cell cycle of germinating barley seeds.     

Reparative DNA synthesis in the lymphocytes of rats exposed to potassium bichromate and manganese chloride in vivo

The intensity of DNA repair synthesis was studied in rat lymphocytes subjected to the treatment in vivo by various concentrations of potassium bichromate and manganese chloride. It was found that manganese chloride has a direct effect on DNA, causing its damage whereas potassium bichromate has an indirect influence on DNA inhibiting its repair synthesis.     

Killing of Chromium-Damaged Cells by Mismatch Repair and its Relevance to Carcinogenesis

Hexavalent chromium compounds are widespread environmental contaminants that are well recognized as human carcinogens and potent respiratory toxicants. Intracellular metabolism of chromium(VI) leads to the production of numerous chromium-DNA adducts that are primarily formed at the phosphate groups. The mechanism of toxicity of these DNA modifications in human cells has been uncertain for a long time because chromium and other phosphate-based adducts did not block DNA replication with purified polymerases. Our recent studies identified mismatch repair proteins as activators of toxic responses to chromium-DNA damage, which resolved an apparent discrepancy in genotoxic activity of chromium adducts in cells and in vitro. The discovered mechanism of toxicity provided the basis for a novel model of chromium carcinogenesis based on the selection of resistant clones that lack mismatch repair and progress to cancer due to high levels of spontaneous mutagenesis.     

Repair replication of opossum lymphocyte DNA: effect of compounds that bind to DNA

Opossum lymphocytes were used for studies of DNA repair. Several compounds were assessed for their capacity to induce repair. Specially interesting was the fact that some intercalators (proflavin, ICR-170, quinacrine and acridine orange) did induce repair, as determined by [³H]thymidine incorporation in the presence of hydroxyurea, CsCl density gradient centrifugation of bromodeoxyuridine-containing DNA and autoradiographically detected unscheduled DNA synthesis.A comparison of the inhibitory effect of several chemicals on DNA replication and DNA repair was also carried out. In this study, repair synthesis was induced by UV irradiation. For most of the compounds, the concentration necessary to inhibit 50% of DNA replication or DNA repair was similar. The most notable exception was cycloheximide which inhibited replication much more effectively than repair. None of the compounds used in this study was found to specifically inhibit repair synthesis.Inhibition of DNA replication and DNA repair was a general effect exhibited by the compounds which bind to DNA. However, only some of these compounds were able to induce repair. As most of these compounds were mutagens it was concluded that the inhibitory effect could be more relevant to mutagenesis that the repair-induction effect.     

2',3'-Dideoxy-3'-aminonucleoside 5'-triphosphates inhibit the repair of DNA in rat liver chromatin

2',3'-Dideoxy-3'-aminonucleoside 5'-triphosphates are shown to be strong inhibitors of repair DNA synthesis in gamma-irradiated rat liver chromatin. The activity of these compounds is comparable with that of the most effective inhibitor of the DNA polymerase beta-catalyzed repair DNA synthesis.     

Current aspects in metal genotoxicity

While carcinogenic metal ions are mostly non-mutagenic in bacteria, different types of cellular damage have been observed in mammalian cells, which may account for their carcinogenic potential. Two modes of action seem to be predominant: the induction of oxidative DNA damage, best established for chromium compounds, and the interaction with DNA repair processes, leading to an enhancement of genotoxicity in combination with a variety of DNA damaging agents. In the case of Cd(II), Ni(II), Co(II), Pb(II) and As(III), DNA repair processes are disturbed at low, non-cytotoxic concentrations of the respective metal compounds. Even though different steps in DNA repair are affected by the diverse metals, one common mechanism might be the competition with essential metal ions.     

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.     

DNA damage excision repair in microplate wells with chemiluminescence detection: development and perspectives

The development of in vitro repair assays with human cell-free extracts led to new insights on the mechanism of excision of DNA damage which consists of incision/excision and repair synthesis/ligation. We have adapted the repair synthesis reaction with cells extracts incubated with damaged plasmid DNA performed in liquid phase to solid phase by DNA adsorption into microplate wells. Since cells extracts are repair competent in base excision and nucleotide excision repair, all types of substrate DNA lesions were detected with chemiluminescence measurement after incorporation of biotin-deoxynucleotide during the repair synthesis step. Derivatives of our initial 3D-assay (DNA damage detection) have been set up to: i) screen antioxidative compounds and NER inhibitors; ii) capture genomic DNA (3D(Cell)-assay) that allows detection of alkylated base and consequently determines the kinetics of the cellular repair; and iii) immunodetect the repair proteins in an ELISA reaction (3D(Rec)-assay). The 3D derived assays are presented and discussed.     

Changing capacity for DNA excision repair in mouse embryonic cells in vitro

Capacity for excision repair of ultraviolet radiation damage to DNA in primary cultures of mouse embryonic cells is dependent on the gestational stage and the duration of in vitro growth. Fibroblasts of mouse embryos at 13–15 days gestation excise thymine dimers and perform unscheduled DNA synthesis after ultraviolet radiation. After several successive transfers in vitro, concomitantly with a pronounced reduction in growth rate, ability for excision repair decreases. DNA repair capacity is impaired in cells obtained from embryos at late stages of development (17–19 days gestation). Experiments with epithelial kidney cells from 5-day-old mice indicate that capacity for excision repair may depend on cell type and its origin.     

Comparison of the action of powerful pulses of ultrashort UV on the DNA replication and transcription functions in proliferating and resting HeLa cells

It was established that the response of proliferating and resting HeLa cells to irradiation with single powerful UV-pulses (lambda = 266 nm, the pulse duration 3.10(-11) sec) is different. In the proliferating cells, the rate of DNA synthesis is markedly changed (accelerated or retarded) while the rate of RNA synthesis changes slightly. In the resting cells, RNA synthesis is stimulated while DNA synthesis remains at the control level. In all cases, the processes observed depend upon the intensity and the number of pulses.     

2′,3′-Dideoxy-3′-aminonucleoside 5′-triphosphates inhibit the repair of DNA in rat liver chromatin

2′,3′-Dideoxy-3′-aminonucleoside 5′-triphosphates are shown to be strong inhibitors of repair DNA synthesis in γ-irradiated rat liver chromatin. The activity of these compounds is comparable with that of the most effective inhibitor of the DNA polymerase β-catalyzed repair DNA synthesis.     

Inhibition of O6-alkylguanine-DNA-alkyltransferase by metals

The activity of the DNA-repair protein O6-alkylguanine-DNA-alkyltransferase was found to be strongly inhibited by a number of metal ions. Cd2+ was the most active followed by Cu2+, Hg2+, Zn2+ and Ag2. This inhibition is likely to result from the interaction of the metals with the cysteine-acceptor residue on the protein since the inhibition was reduced by increasing the concentration of dithiothreitol in the assay buffer. These results raise the possibility that exposure to Cd2+ could increase the mutagenicity and carcinogenicity of alkylating agents by retarding the rate of repair of alkylated DNA. However, other metals or metallic compounds which are known to be carcinogenic (such as compounds containing arsenic, lead, nickel or chromium) did not interfere with DNA repair by this protein.     

Weakening of the ultraviolet ray-induced unscheduled DNA synthesis in human lymphocytes in extreme old age

The intensity of unscheduled DNA synthesis was studied in UV-irradiated (10--15 J/m2) peripheral blood lymphocytes of 80--90 years old persons. In these extreme old age persons, reparative DNA synthesis was found sufficiently reduced in comparison with that in middle aged (20--43 years old) ones. The role of DNA repair processes in ageing is under discussion.     

Involvement of DNA polymerase delta in DNA repair synthesis in human fibroblasts at late times after ultraviolet irradiation

DNA repair synthesis following UV irradiation of confluent human fibroblasts has a biphasic time course with an early phase of rapid nucleotide incorporation and a late phase of much slower nucleotide incorporation. The biphasic nature of this curve suggests that two distinct DNA repair systems may be operative. Previous studies have specifically implicated DNA polymerase delta as the enzyme involved in DNA repair synthesis occurring immediately after UV damage. In this paper, we describe studies of DNA polymerase involvement in DNA repair synthesis in confluent human fibroblasts at late times after UV irradiation. Late UV-induced DNA repair synthesis in both intact and permeable cells was found to be inhibited by aphidicolin, indicating the involvement of one of the aphidicolin-sensitive DNA polymerases, alpha or delta. In permeable cells, the process was further analyzed by using the nucleotide analogue (butylphenyl)-2'-deoxyguanosine 5'-triphosphate, which inhibits DNA polymerase alpha several hundred times more strongly than it inhibits DNA polymerase delta. The (butylphenyl)-2'-deoxyguanosine 5'-triphosphate inhibition curve for late UV-induced repair synthesis was very similar to that for polymerase delta. It appears that repair synthesis at late times after UV irradiation, like repair synthesis at early times, is mediated by DNA polymerase delta.     

DNA repair synthesis induced by N-hydroxyurea, acetohydroxamic acid, and N-hydroxyurethane in primary rat hepatocyte cultures: comparative evaluation using the autoradiographic and the bromodeoxyuridine density-shift method

N-Hydroxyurea and two structurally related compounds, acetohydroxamic acid and N-hydroxyurethane, were investigated for their potential to induce DNA repair synthesis in primary rat hepatocyte cultures. Repair was determined as repair replication by means of the bromodeoxyuridine density-shift method and, in the same cell preparations, as unscheduled DNA synthesis (UDS) by autoradiography. For all 3 compounds, a clear concentration-dependent induction of DNA repair replication could be demonstrated. Interpretation of the UDS data, however, depended on the mode whereby the results were evaluated. Expression of the results as net grains per nucleus after subtraction of cytoplasmic from nuclear grain counts yielded statistically significant increases over the control values for all compounds. In contrast, no significant changes of the nuclear labeling were obtained when nuclear and cytoplasmic grain counts were plotted separately. These findings demonstrate that the two modes to present UDS data may lead to different conclusions, a consequence of the uncertainty regarding the origin and importance of the cytoplasmic background. The observation that both hydroxyurea and the structurally related compounds acetohydroxamic acid and N-hydroxyurethane induce DNA repair in primary hepatocyte cultures suggests that metabolism-dependent genotoxicity may be a common property of aliphatic hydroxamic acids.     

Enhancement by transition metals of unscheduled DNA synthesis induced by isoniazid and related hydrazines in cultured normal and xeroderma pigmentosum human cells

In combination with transition metals (Mn(II), Cu(II), and Fe(III)), isoniazid and related hydrazine compounds induced unscheduled DNA synthesis (DNA repair) in cultured human fibroblasts. Manganese at 10(-5) and 10(-4) M strongly enhanced DNA repair induced by isoniazid, iproniazid, nialamide and hydrazine. Peak levels of DNA repair occurred at 5 x 10(-4)--10(-3) M of the 4 hydrazine compounds. Copper caused less enhancement of DNA repair while iron had no detectable effect. Without added metal, unscheduled DNA synthesis was not observed in cells treated with any of the 4 freshly-prepared hydrazine compounds. However, following preincubation in medium for 6--12 h, isoniazid alone at high concentrations (10(-2) M--10(-1) M) induced DNA repair. With isoniazid/manganese mixtures, preincubation did not further enhance DNA repair except at low concentrations of isoniazid (2--5 x 10(-4) M). Catalase reduced the DNA damage caused by preincubated isoniazid and by the isoniazid/metal mixtures. Exposure of repair-deficient xeroderma pigmentosum cells to isoniazid plus manganese resulted in a DNA-repair profile similar to that of normal cells. The results are consistent with hydrogen peroxide being a critical intermediate for the production of free radicals which cause the observed DNA damage.     

Repair and subsequent fragmentation of deoxyribonucleic acid in ultraviolet-irradiated Bacillus subtilis recA.

Cells of Bacillus subtilis recA1 are sensitive to irradiation with ultraviolet light. Evidence is presented here that these cells are not defective in ultraviolet light-induced incision of deoxyribonucleic acid (DNA) or repair DNA synthesis. Ligation of DNA at repair sites appears to occur, but the DNA is subsequently fragmented, apparently at sites of previous repair synthesis. It is hypothesized that the defect in DNA repair leads to host-specific restriction at repaired sites because of a defect in either the structure of the repaired region or specificity of the restriction/modification system.     

The effect of deoxyadenosine plus deoxycoformycin on replicative and repair synthesis of DNA in human lymphoblasts and isolated nuclei

Deoxyadenosine plus deoxycoformycin (dCf) causes increased DNA breaks in lymphoid cells. This study explored the possible inhibition of repair synthesis of DNA by dAdo plus dCf as a cause of DNA breakage. It was shown that DNA breaks accumulated in a human T-lymphoblast cell line, CCRF-CEM, following incubation with dAdo plus dCf and were not fully repaired 20 h after their removal. Analysis of the density distribution of radiolabeled DNA on alkaline CsCl gradient showed that incubation of CCRF-CEM cells with dAdo plus dCf caused inhibition of semiconservative, but not repair synthesis of DNA. Semiconservative synthesis of DNA was also inhibited in CCRF-CEM nuclei isolated from cells pretreated with dAdo and dCf, suggesting damage to DNA replicative machinery. However, no such inhibition was observed in the nuclei of a similarly treated CCRF-CEM mutant that was deficient in adenosine kinase and deoxycytidine kinase. This suggests that dAdo must be phosphorylated in intact cells to exert its effect. Using [3H]dTTP incorporation in isolated CCRF-CEM nuclei to measure DNA synthesis, it was found that a high concentration (greater than 100 microM) of dATP inhibits semiconservative but not repair synthesis of DNA. The present studies thus indicate that accumulation of DNA strand breaks induced by dAdo plus dCf is not the consequence of inhibition of repair DNA synthesis. This implies the mechanism may involve perturbation of DNA ligation or activation of a certain process which causes DNA strand breaks. In addition, dATP may interfere with some steps of semiconservative DNA synthesis, but not the repair synthesis of DNA.     

Multiple subphases of DNA repair and poly (ADP-ribose) synthesis in Chinese hamster ovary (CHO-K1) cells

The two types of DNA synthesis as well as poly(ADP-ribose) biosynthesis were measured simultaneously in synchronized intact populations of CHO cells throughout the duration of S phase. Naturally occurring DNA fragmentation was detected by random primed oligonucleotide synthesis (ROPS assay). Fractions of synchronous cell populations were obtained by counterflow centrifugal elutriation. By gradually increasing the resolution of centrifugal elutriation multiple non-overlapping repair and replication peaks were obtained. The elutriation profile of DNA repair peaks corresponded to the DNA fragmentation pattern measured by ROPS assay. The number and position of poly(ADP-ribose) peaks during S phase resembled those seen in the DNA replication profile. Our results indicate that PAR synthesis is coupled to DNA replication serving the purpose of genomic stability.     

Excision repair of DNA in nuclear extracts from the yeast Saccharomyces cerevisiae.

Excision repair of DNA is an important cellular response to DNA damage caused by a broad spectrum of physical and chemical agents. We have established a cell-free system in which damage-specific DNA repair synthesis can be demonstrated in vitro with nuclear extracts from the yeast Saccharomyces cerevisiae. Repair synthesis of UV-irradiated plasmid DNA was observed in a radiation dose-dependent manner and was unaffected by mutations in the RAD1, RAD2, RAD3, RAD4, RAD10, or APN1 genes. DNA damaged with cis-platin was not recognized as a substrate for repair synthesis. Further examination of the repair synthesis observed with UV-irradiated DNA revealed that it is dependent on the presence of endonuclease III-sensitive lesions in DNA, but not pyrimidine dimers. These observations suggest that the repair synthesis observed in yeast nuclear extracts reflects base excision repair of DNA. Our data indicate that the patch size of this repair synthesis is at least seven nucleotides. This system is expected to facilitate the identification of specific gene products which participate in base excision repair in yeast.