Mechanisms of DNA repair and the structure of repaired sites in the presence of polymerase inhibitors.
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Anti-Bacterial Agents↗
Biomedical subjects
J E Cleaver
Publications and source records attributed to J E Cleaver.
Inhibition of X-ray- and ultraviolet light-induced transformation in vitro by modifiers of poly(ADP-ribose) synthesis.
Neoplastic transformation in vitro of hamster embryo cells and mouse C3H 10T1/2 cells by X rays and ultraviolet light was suppressed by benzamide or 3-aminobenzamide, agents which inhibit poly(ADP-ribose) polymerization. Suppression was observed under conditions in which the inhibitors reduce poly(ADP-ribose) polymerization by about 75% and increase sister chromatid exchange frequencies, but have no influence on repair of X-ray and uv damage and reportedly have no detectable side effects on nucleotide precursor metabolism. These findings suggest that the mechanisms regulating neoplastic transformation differ from those regulating mutagenesis and sister chromatid exchanges and are mediated via alterations in poly(ADP-ribosylation), causing changes in gene control and expression.
Animals↗
Inhibition by hyperthermia of repair synthesis and chromatin reassembly of ultraviolet-induced damage to DNA.
We have investigated the effects of hyperthermia treatment on sequential steps of the repair of UV-induced DNA damage in HeLa cells. DNA repair synthesis was inhibited by 40% after 15 min of hyperthermia treatment at 45 degrees C; greater inhibition of repair synthesis occurred with prolonged incubation at 45 degrees C. Enzymatic digestion of repair-labeled DNA with Exonuclease III indicated that once DNA repair was initiated, the DNA repair patch was synthesized to completion and that ligation of the DNA repair patch occurred. Thus the observed inhibition of UV-induced DNA repair synthesis by hyperthermia treatment may be the result of inhibition of enzymes involved in the initiating step(s) of DNA repair. DNA repair patches synthesized in UV-irradiated cells labeled at 37 degrees C with [3H]Thd were 2.2-fold more sensitive to micrococcal nuclease digestion than was parental DNA; if the length of the labeling period was prolonged, the nuclease sensitivity of the repair patch synthesized approached that of the parental DNA. DNA repair patches synthesized at 45 degrees C, however, remained sensitive to micrococcal nuclease digestion even after long labeling periods, indicating that heat treatment inhibits the reassembly of the DNA repair patch into nucleosomal structures.
Chromatin↗
Restriction enzyme cleavage of ultraviolet-damaged simian virus 40 and pBR322 DNA.
Cleavage of specific DNA sequences by the restriction enzymes EcoRI, HindIII and TaqI was prevented when the DNA was irradiated with ultraviolet light. Most of the effects were attributed to cyclobutane pyrimidine dimers in the recognition sequences; the effectiveness of irradiation was directly proportional to the number of potential dimer sites in the DNA. Combining EcoRI with dimer-specific endonuclease digestion revealed that pyrimidine dimers blocked cleavage within one base-pair on the strand opposite to the dimer but did not block cleavage three to four base-pairs away on the same strand. These are the probable limits for the range of influence of pyrimidine dimers along the DNA, at least for this enzyme. The effect of irradiation on cleavage by TaqI seemed far greater than expected for the cyclobutane dimer yield, possibly because of effects from photoproducts flanking the tetranucleotide recognition sequence and the effect of non-cyclobutane (6-4)pyrimidine photoproducts involving adjacent T and C bases.
DNA Restriction Enzymes↗
Differences in the regulation by poly(ADP-ribose) of repair of DNA damage from alkylating agents and ultraviolet light according to cell type.
Inhibition of poly(ADP-ribose) synthesis by 3-aminobenzamide in various human and hamster cells influenced the responses to DNA damage from methyl methanesulfonate, but not from ultraviolet light. After exposure to methyl methanesulfonate, 3-aminobenzamide increased the strand break frequency in all cell types studied, but only stimulated repair replication in lymphoid and HeLa cells, suggesting these are independent effects. 3-Aminobenzamide also inhibited the pathway for de novo synthesis of DNA purines, suggesting that some of its effects may be due to disturbance of precursor pathways and irrelevant to the role of poly(ADP-ribose) in repair. Previous claims that 3-aminobenzamide stimulates repair synthesis after exposure to UV light are probably artifacts, because the stimulations are only observed in lymphocytes in the presence of a high concentration of hydroxyurea that itself inhibits repair. The initial inhibition of semiconservative DNA synthesis and the excision of the major alkylation products and pyrimidine dimers were unaffected by 3-aminobenzamide. In general poly(ADP-ribose) synthesis appears to be uniquely involved in regulating the ligation stage of repair of alkylation damage but not ultraviolet damage. By regulating the ligation efficiency, poly(ADP-ribosylation) modulates the dynamic balance between incision and ligation, so as to minimize the frequency of DNA breaks. The ligation stage of repair of UV damage appears different and is not regulated by poly(ADP-ribosylation).
Animals↗
Structure of repaired sites in human DNA synthesized in the presence of inhibitors of DNA polymerases alpha and beta in human fibroblasts.
Excision repair of ultraviolet damage in human fibroblasts was partially inhibited by drugs that block DNA polymerases alpha or beta (cytosine arabinoside, aphidicolin and dideoxythymidine) causing a reduction in unscheduled synthesis and an accumulation of single-strand breaks. The strand breaks accumulated in the presence of aphidicolin could be resealed within 30 min after removal of the drug, but those accumulated by cytosine arabinoside took many hours. Digestion of repaired DNA with exonuclease III or S1 nuclease revealed that even the highest concentration of polymerase inhibitors, singly or in combination, that produced maximal accumulation of single-strand breaks only blocked 37-86% of repair sites. Use of single-strand break frequencies to measure the number of repair events can therefore be in error by as much as a factor of 3. The blocked patches with free 3'OH termini were, on average, 22% of normal length, corresponding to between 6 and 17 bases (assuming a normal patch of 25-75 bases in length). Patches that remained unsealed in vivo were also resistant to sealing by T4 ligase in vitro. The data are more consistent with a mechanism of repair in which long single-strand gaps are first made by excision enzymes and subsequently filled in by DNA polymerase alpha. Strand displacement or nick translation mechanisms seem unlikely.
Aphidicolin↗
Replicon size and excision repair as factors in the inhibition and recovery of DNA synthesis from ultraviolet damage.
Initiation of DNA replication and chain growth, analyzed by alkaline sucrose gradient sedimentation, was interrupted to different extents in different cell types by irradiation with ultraviolet light. Within the first hour of irradiation DNA replication was reduced in a manner that depended on the average number of lesions per replicating unit (replicon). At low numbers of lesions per replicon, inhibition of replicon initiation was the predominant response; at higher numbers of lesions per replicon, blockage of chain growth was also observed. After irradiation with a dose that initially blocked chain growth, the rate at which cells recovered their ability to synthesize increasingly more and larger size DNA was a function both of replicon size and of excision repair capacity. Cells with small replicons recovered more rapidly than cells with large replicons, and excision repair-deficient cells recovered less rapidly than excision-competent cells. These observations indicate that excision repair capacity and replicon size play major roles in the response of DNA replication to ultraviolet damage.
Animals↗
DNA excision repair in permeable human fibroblasts.
U.v. irradiation of confluent human fibroblasts activated DNA repair, aspects of which were characterized in the cells after they were permeabilized. Incubation of intact cells for 20 min between irradiation and harvesting was necessary to obtain a maximum rate of reparative DNA synthesis. Cells harvested immediately after irradiation before repair was initiated displayed only a small stimulation of DNA synthesis, indicating that permeable cells have a reduced capacity to recognize pyrimidine dimers and activate repair. The distribution of sizes of DNA strands labeled during 10 min of reparative DNA synthesis resembled that of parental DNA. However, during a 60-min incubation of permeable cells at 37 degrees C, parental DNA and DNA labeled by reparative DNA synthesis were both cleaved to smaller sizes. Cleavage also occurred in unirradiated cells, indicating that endogenous nuclease was active during incubation. Repair patches synthesized in permeable cells displayed increased sensitivity to digestion by micrococcal nuclease. However, the change in sensitivity during a chase with unlabeled DNA precursors was small, suggesting that reassembly of nucleosome structure at sites of repair was impaired. To examine whether this deficiency was due to a preponderance of incomplete or unligated repair patches, 3H-labeled (repaired) DNA was purified, then digested with exonuclease III and nuclease S1 to probe for free 3' ends and single-stranded regions. About 85% of the [3H]DNA synthesized during a 10-min pulse resisted digestion, suggesting that a major fraction of the repair patches that were filled were also ligated. U.v. light-activated DNA synthesis in permeable cells, therefore, appears to represent the continuation of reparative gap-filling at sites of excision repair activated within intact cells. Gap-filling and ligation were comparatively efficient processes in permeable cells, whereas activation of reparative DNA synthesis and reassembly of native chromatin structure upon completion of repair were not.
Cell Membrane Permeability↗
Effect of 3-aminobenzamide on the rate of ligation during repair of alkylated DNA in human fibroblasts.
3-Aminobenzamide, an inhibitor of polyadenosine diphosphoribose polymerase, produced rapid reversible changes in single-strand break frequencies in DNA from primary human fibroblasts damaged by alkylating agents, but it did not cause such changes in the DNA of cells damaged by ultraviolet light. The increase in single-strand peak frequencies was not due to an accumulation of blocked repair sites, such as occurs with DNA polymerase inhibitors, but to a delay in the rejoining of induced breaks. 3-Aminobenzamide increases the net break frequency that results from a dynamic balance between excision and ligation. This balance appears to be regulated at the ligation step by adenosine diphosphate ribosylation, which is rapidly altered by addition or removal of 3-aminobenzamide. The rapidity with which strand break frequencies change in the presence of 3-aminobenzamide implies that individual strand breaks resulting from excision at any time after exposure have a lifetime of no more than about 30 min in the cell.
Benzamides↗
Critical biochemical and regulatory events in malignant transformation in vitro.
Oncogenic transformation of hamster embryo cells and mouse C3H 10T1/2 can be modified by a variety of agents and conditions which alter events at early stages of initiation and at later stages of promotion. Inhibition of poly(ADP-ribose) synthesis by low concentrations of benzamide and 3-aminobenzamide inhibits the induction of transformation by ultraviolet light, X-rays, and chemical carcinogens as well as inhibiting the enhancement of transformation by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). The suppression of transformation by the benzamides is observed under conditions where the inhibitors reduce poly(ADP-polymerization) by about 75%, have no influence on damage induced by X-rays or alkylating chemicals and enhance sister chromatid exchanges. A modification of the physiological state of the cells by rendering them hypothyroid also results in an inhibition of transformation by radiation and chemical carcinogens and a suppression of promotion by teleocidin and TPA. When thyroid hormone is added to the medium radiogenic transformation and its dramatic enhancement by the tumor promoters is observed, with teleocidin being over 100 times as effective as a promotor as TPA. Our results suggest that mechanisms regulating initiation and promotion are associated with alterations in poly(ADP-ribosylation), causing changes in gene control and expression and may differ from those associated with the induction of sister chromatid exchanges. The results also suggest that genetic events taking place in both the early events (initiation) and late events (promotion) in malignant transformation are highly dependent on the presence of thyroid hormones.
Animals↗
Poly(ADP-ribose): spectator or participant in excision repair of DNA damage.
Inhibition of poly(ADP-ribose) synthesis by 3-aminobenzamide in various human and hamster cell types influenced the responses to DNA damage from methyl methanesulfonate (MMS), but not from UV light. Excision of the major alkylation products and pyrimidine dimers was unaffected by 3-aminobenzamide. After exposure of cells to methyl methanesulfonate, 3-aminobenzamide increased the strand break frequency in all cell types studied, but stimulated repair replication only in lymphoid and HeLa cells, suggesting these are independent effects. 3-Aminobenzamide also inhibited the pathway for de novo synthesis of DNA purines, suggesting that some of its effects, particularly on repair replication, may be due to disturbance of precursor pathways. 3-Aminobenzamide stimulated sister chromatid exchange formation and mutagenesis but inhibited transformation, suggesting that some of these endpoints involve ADP-ribosylation by ways other than repair. Poly(ADP-ribose) synthesis appears to regulate the ligation stage of repair of alkylation damage by modulating a dynamic balance between incision and ligation, so as to minimize the frequency of DNA breaks.
Animals↗
Enzyme digestion of intermediates of excision repair in human cells irradiated with ultraviolet light.
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Cell Line↗
Comparison of nucleoid and alkaline sucrose gradients in the analysis of inhibitors of DNA repair in human fibroblasts.
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Aphidicolin↗
Restriction enzyme cleavage of ultraviolet-damaged DNA.
SV40 and pBR322 DNAs damaged by ultraviolet light were cleaved abnormally by several restriction enzymes because of damage to pyrimidines in the recognition sequences. The use of a tandemly duplicated plasmid provided a particularly sensitive target molecule for detecting pyrimidine dimers and other possible photoproducts. The relative efficiency with which cleavage was blocked (HindIII greater than TaqI greater than EcoRI greater than BamI greater than SalI much greater than Hha I, Hae III) corresponds approximately to the relative frequency of pyrimidine dimer formation in the recognition sequences, but at a slightly higher frequency in potential sites for the non-cyclobutane T-C product. The pyrimidine dimers appear to have a range of influence that extends 1 to 3 basepairs along the DNA molecule. These effects provide clues to the way DNA damage from mutagens and carcinogens can interfere with specific enzyme-DNA interactions.
Base Sequence↗
Rapid complementation method for classifying excision repair-defective xeroderma pigmentosum cell strains.
A rapid method has been developed that permits demonstration of complementation between different cell strains from ultraviolet-sensitive xeroderma pigmentosum patients. Combining polyethylene glycol-mediated cell fusion with low doses of ultraviolet light to eliminate unfused sensitive cells, the method permits assignment of cell strains to complementation groups by visual inspection, avoiding use of laborious methods involving autoradiography. This method can be augmented by measuring DNA repair synthesis, which shows large quantitative differences between fusions that result in complementation and those that do not.
Cell Line↗
DNA repair assays as tests for environmental mutagens. A report of the U.S. EPA Gene-Tox Program.
A literature review was undertaken to determine the usefulness of DNA repair assays, other than unscheduled DNA synthesis, as screening techniques for mutagenic carcinogens. 92 reports were found to contain useful data for 49 chemicals using 6 techniques, namely, (1) cesium chloride equilibrium density gradients to study repair replication, (2) benzoylated naphthoylated diethylaminoethyl cellulose columns to study repair replication, (3) 313-nm irradiation of DNA containing bromodeoxyuridine to study repair replication, (4) alkaline elution to study repair of single-strand breaks and crosslinks, (5) alkaline sucrose gradients to study repair of single-strand breaks, and (6) direct assays for removal of adducts from DNA. Almost all of the 49 chemicals studied were known mutagens or carcinogens and/or known inducers of DNA repair, 9 compounds failed to elicit DNA repair by at least 1 assay technique, and at least 3 of these were not tested by the most appropriate and sensitive method. Nevertheless, although valid for studying repair phenomena in eukaryotic cells, these assays are not considered useful for screening. They are time-consuming, expensive, and/or require highly specialized skills. Despite the high frequency of positive reports, it is obvious from the literature that repair assays will fail to detect, or will detect with low efficiency, those agents whose main action is either intercalation or induction of strand breaks. For these and other reasons, DNA repair as a basis for screening for mutagenic carcinogens is not considered to be a useful concept.
Bromodeoxyuridine↗
3-Aminobenzamide synergistically increases sister-chromatid exchanges in cells exposed to methyl methanesulfonate but not to ultraviolet light.
3-Aminobenzamide, an inhibitor of poly(ADP-ribose) synthesis, increased baseline sister-chromatid exchange (SCE) frequencies and acted synergistically with the alkylating agent methyl methanesulfonate to induce exchanges in Chinese hamster ovary and SV40-transformed human (GM637) cells. In contrast, 3-aminobenzamide did not affect the frequency of ultraviolet light-induced SCEs. Our data suggest that, in these 2 cell types, synthesis of poly(ADP-ribose) is more important in damage and repair after exposure to an alkylating agent than after exposure to ultraviolet light.
Animals↗
Specificity and completeness of inhibition of DNA repair by novobiocin and aphidicolin.
Novobiocin and aphidicolin were both potent inhibitors of excision repair of u.v.-induced damage to DNA in human embryonic fibroblasts, and both also inhibited semiconservative DNA replication even more strongly. The mechanism of action of these two drugs is, however, different. Novobiocin inhibited repair replication without accumulating single-strand breaks, but aphidicolin inhibited repair replication with the accumulation of numerous single-strand breaks. Novobiocin appears to inhibit repair at an earlier stage than aphidicolin, which may indicate that DNA topoisomerases play a role in eukaryotic DNA repair. Digestion of DNA by exonuclease III indicated that repair patches in novobiocin-treated cells contained no excess 3'OH termini, whereas up to 40% of the repaired DNA in aphidicolin-treated cells had free 3'OH termini. Therefore, although aphidicolin resulted in the accumulation of single-strand breaks, many of the repair events escaped inhibition and the number of breaks is an underestimate of the true number of repair events.
Antibiotics, Antineoplastic↗