Base excision repair is efficient in cells lacking poly(ADP-ribose) polymerase 1

Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that is activated by binding to DNA breaks induced by ionizing radiation or through repair of altered bases in DNA by base excision repair. Mice lacking PARP-1 and, in certain cases, the cells derived from these mice exhibit hypersensitivity to ionizing radiation and alkylating agents. In this study we investigated base excision repair in cells lacking PARP-1 in order to elucidate whether their augmented sensitivity to DNA damaging agents is due to an impairment of the base excision repair pathway. Extracts prepared from wild-type cells or cells lacking PARP-1 were similar in their ability to repair plasmid DNA damaged by either X-rays (single-strand DNA breaks) or by N-methyl-N′-nitro-N-nitrosoguanidine (methylated bases). In addition, we demonstrated in vivo that PARP-1-deficient cells treated with N-methyl-N′-nitro-N-nitrosoguanidine repaired their genomic DNA as efficiently as wild-type cells. Therefore, we conclude that cells lacking PARP-1 have a normal capacity to repair single-strand DNA breaks inflicted by X-irradiation or breaks formed during the repair of modified bases. We propose that the hypersensitivity of PARP-1 null mutant cells to γ-irradiation and alkylating agents is not directly due to a defect in DNA repair itself, but rather results from greatly reduced poly(ADP-ribose) formation during base excision repair in these cells.     

The role of DNA polymerases in DNA repair in Escherichia coli: DNA polymerase I-dependent repair following chemical alkylation of permeabilized bacteria.

Many mutagens and carcinogens damage DNA and elicit repair synthesis in cells. In the present study we report that alkylation of the DNA of Escherichia coli that have been made permeable to nucleotides by toluene treatment results in the expression of a DNA polymerase I-directed repair synthesis. The advantage of the system described here is that it permits measurement of only DNA polymerase I-directed repair synthesis and serves as a simple, rapid method for determining the ability of a given chemical to elicit “excision-repair” in bacteria.DNA ligation is intentionally prevented in our system by addition of the inhibitor nicotinamide mononucleotide. In the absence of DNA ligase activity, nick translation is extensive and an “exaggerated” repair synthesis occurs. This amplification of repair synthesis is unique for DNA polymerase I since it is not observed in mutant cells deficient in this polymerase. DNA ligase apparently controls the extent of nucleotide replacement by this repair enzyme through its ability to rejoin “nicks” thereby terminating the DNA elongation process.The nitrosoamides N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea, as well as the nitrosoamidines N-methyl-N′-nitro-N-nitrosoguanidine and N-ethyl-N′-nitro-N-nitrosoguanidine, elicit DNA polymerase I-directed repair synthesis. Methyl methanesulphonate is especially potent in this regard, while its ethyl derivative, ethyl methanesulphonate, is a poor inducer of DNA polymerase I activity in permeabilized cells.     

Increased sensitivity of xeroderma pigmentosum cells to some chemical carcinogens and mutagens

The clone-forming capacity and level of DNA repair was examined on normal human cells and repair-deficient Xeroderma pigmentosum (XP) fibroblasts exposed to various chemical carcinogens and mutagens.The cultured fibroblasts were treated for 90 min with the carcinogenic and mutagenic 4-nitroquinoline 1-oxide (4NQO), 4-hydroxyaminoquinoline 1-oxide (4HAQO), 2-methyl-4-nitroquinoline 1-oxide (2-Me-4NQO), 3-methyl-4-nitropyridine 1-oxide 3-Me-4NPO) and the non-carcinogenic 6-nitroquinoline 1-oxide (6NQO). The response of the cells to the N-oxides was compared to that induced by the mutagen and carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and UV-irradiation.The XP cells showed (1) a reduced level of DNA repair synthesis when exposed to various carcinogenic N-oxides, (2) no unscheduled DNA synthesis following 6NQO and (3) a normal degree of DNA repair synthesis after treatment with MNNG.When the clone-forming capacity was examined the XP cells exhibited (1) a higher increased sensitivity to the various carcinogenic N-oxides, (2) no reduction in the clone formation following 6NQO and (3) a sensitivity virtually comparable to that of normal cells after treatment with MNNG.The results suggest a link between extent of DNA damage, level of DNA repair and degree of sensitivity in human cells exposed to various chemical carcinogens and which induce DNA alterations that cannot be repaired by DNA repair synthesis.     

Differential sensitivity of DNA replication and repair in permeable Escherichia coli exposed to various monofunctional methylating or ethylating agents.

Escherichia coli cells made permeable to deoxynucleoside triphosphates by brief treatment with toluene (permeablized) were used to measure the effect of the following chemical alkylating agents on either DNA replication or DNA repair synthesis: methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and N-ethyl-N′-nitro-N-nitrosoguanidine (ENNG). Replication of DNA in this pseudo-in vivo system was completely inhibited 10–15 min after exposure to MMS at concentrations of 5 mM or higher or to MNU or MNNG at concentrations of 1 mM or higher. The ethyl derivatives of the alkylating agents were less inhibitory than their corresponding methyl derivatives, and inhibition of DNA replication occurred in the following order: EMS < ENNG < ENU. Maximum inhibition of DNA replication by all of the alkylating agents tested except EMS occurred at a concentration of 20 mM or lower. The extent of replication in cells exposed to EMS continued to decrease with concentrations of EMS up to 100 mM (the highest concentration tested).The experiments in which the inhibition of DNA replication by MMS, MNU, or MNNG was measured were repeated under similar assay conditions except that a density label was included and the DNA was banded in CsCl gradients. The bulk of the newly synthesized DNA from the untreated cells was found to be of the replicative (semi-conservative) type. The amount of replicative DNA decreased with increasing concentration of methylating agent in a manner similar to that observed in the incorporation experiments.Polymerase I (Pol I)-directed DNA repair synthesis induced by X-irradiation of permeablized cells was assayed under conditions that blocked the activity of DNA polymerases II and III. Exposure of cells to MNNG or ENNG at a concentration of 20 mM resulted in reductions in Pol I activity of 40 and 30%, respectively, compared with untreated controls. ENU was slightly inhibitory to Pol I activity, while MMS, EMS, and MNU all caused some enhancement of Pol I activity.These data show that DNA replication in a pseudo-in vivo bacterial system is particularly sensitive to the actions of known chemical mutagens, whereas DNA repair carried out by the Pol I repair enzyme is much less sensitive and in some cases apparently unaffected by such treatment. Possible mechanisms for this differential effect on DNA metabolism and its correlation with current theories of chemically induced mutagenesis and carcinogenesis are discussed.     

Isolation and Characterization of a Bacillus subtilis Mutant with a Defective N-Glycosidase Activity for Uracil-Containing Deoxyribonucleic Acid

Crude cell extracts of Bacillus subtilis 168T exhibit enzyme activity capable of releasing free uracil from phage PBS1 deoxyribonucleic acid (DNA) in the presence of ethylenediaminetetraacetate. By measuring the enzyme activity in 300 clones that emanated from mutagenized cells, we obtained a mutant strain that did not show this N-glycosidase activity. The mutant strain, designated as TKJ6901 (urg-1) exhibited no physiological abnormalities. We observed the intracellular action of the enzyme by following the fate of uracil-containing DNA in cells from wild-type and mutant cultures. When infection with phage PBS1 was allowed in the presence of chloramphenicol, extensive degradation of phage DNA was observed only in the wild-type cells. When bromouracil residues were converted to uracil residues by ultraviolet light irradiation in the presence of cysteamine, the DNA was extensively fragmented in the wild-type cells. These single-strand breaks were rejoined upon postirradiation incubation. In contrast, such fragmentation of the DNA was not observed in the mutant cells, indicating that the uracil residues were not removed from the DNA. This demonstrated that the N-glycosidase activity was involved in the excision of uracil in DNA. A transformation assay with four types of recipient strains with combinations of N-glycosidase and DNA polymerase I deficiencies indicated that DNA polymerase I was involved in the later steps of this base excision repair pathway initiated by the action of the N-glycosidase.     

Assessment of the cellular internalization of thermolytic phosphorothioate DNA oligonucleotide prodrugs

The bioactivity of a CpG-containing phosphorothioate DNA oligonucleotide with thermolytic 2-(N-formyl-N-methylamino)ethyl (fma) thiophosphate groups in mice led us to investigate the parameters affecting the internalization of these thermosensitive DNA prodrugs in various cell lines. Flow cytometry and confocal microscopy analyses indicate that 5′-fluoresceinated fma-phosphorothioate DNA sequences are poorly internalized in Vero, HeLa and GC-2 cells. However, when four fma-thiophosphate groups of a 15-nucleotide long oligothymidylate prodrug are replaced with 3-(N,N-dimethylamino)prop-1-yl thiophosphate functions, internalization of the positively charged prodrug, under physiological conditions, increased fourfold in HeLa and 40-fold in Vero or GC-2 cells. No cytotoxic effects are observed in Vero cells even at an extracellular prodrug concentration of 50 μM over a period of 72 h. Confocal microscopy studies show that internalization of the positively charged oligothymidylate prodrug in Vero cells is time-dependent with early trafficking of the DNA sequence through endosomal vesicles and, eventually, to the nucleus of the cells. Thus, the incorporation of four 3-(N,N-dimethylamino)prop-1-yl thiophosphate groups into thermosentive fma-phosphorothioate DNA prodrugs is an attractive strategy for efficient cellular internalization of these nucleic acid-based drugs for potential therapeutic indications.     

Repair of lesions induced in human liver cell DNA by N-nitroso compounds as measured by the bromodeoxyuridine photolysis method

Chang human liver cells were treated with the carcinogens N-methyl-N′-nitrosoguanidine (MNNG) and nitrosomorpholine (NM). In addition, cells were exposed to the folic acid analog, 2-hydroxy-N¹⁰ nitrosofolic acid. Repair of the damage to DNA was estimated by selective photolysis of BUdR-containing repaired regions with 313 nm radiation. The influence of the co-carcinogen Arlacel A was estimated with the three compounds. Results indicated significant repair synthesis with MNNG- and NM-treated cells. 2-Hydroxy-N¹⁰ nitrosofolic acid elicited no damage to the liver DNA. Arlacel A prevented repair synthesis in cells treated with NM and MNNG.     

A comparison of the DNA binding,cytotoxicity and repair synthesis induced in human fibroblasts by reactive derivatives of aromatic amide carcinogens

The cytotoxicity of three structurally-related direct-acting carcinogens, N-acetoxy-2-acetylaminofluorene, N-acetoxy-2-acetylaminophenanthrene and N-acetoxy-4-acetylaminobiphenyl, was compared in normal cells and in excision repair deficient xeroderma pigmentosum cells (XP12BE). All three proved significantly more cytotoxic to the XP cells than to the normal cells. At equicytoxic levels, substantially more residues were initially bound to the DNA of the normal cells than to the XP cells, suggesting that the former are able to remove a large percentage of the DNA bound residues before these can result in cell death. The ability of these cell strains to remove bound residues from DNA, to incorporate thymidine into parental strands of DNA during repair replication, and to recover from potentially lethal damage if held in the non-replicating, density-inhibited G_o state was compared as a function of dose and time. The XP12BE cells proved virtually incapable of excision repair of DNA damage induced by these carcinogens and of recovery. In contrast, normal cells recovered from the potentially lethal effects of these three compounds and did so at a rate comparable to their rate of removal of bound residues and of repair synthesis. In the excision-deficient XP12BE cells, DNA adducts induced by N-acetoxy-2-acetylaminophenanthrene proved 3- to 6-fold more cytotoxic than adducts induced by the other two carcinogens.     

Differences between diploid and transformed human cells in the incorporation of 5-bromodeoxyuridine into DNA

In diploid human cells, the DNA precursor pool equilibration times for exogenous thymidine are about twice those for the thymidine analogue 5-bromodeoxyuridine (BUdR); in cells that were either transformed chemically or derived from malignant tumours, the pool equilibration times are the same for thymidine and 5-bromodeoxyuridine and are closer in value to the shorter (bromodeoxyuridine) times of the diploid cells. Thymidine, if present in the culture medium with BUdR, is incorporated into DNA preferentially in diploid cells (by 2 or 3 to 1). Discrimination against bromodeoxyuridine is evident within 2 h of incubation of the two precursors with diploid cells, but is not observed even after 24 h in any of the transformed cell lines tested. Experiments were performed to test the effect of inhibitors of the mammalian DNA polymerases alpha (N-ethylmaleimide) and beta (incubation of cells at 45 °C) upon the ability of cells to synthesise DNA and to incorporate thymidine preferentially when present with equimolar BUdR. In diploid cells, overall in vivo DNA synthesis is more sensitive to N-ethylmaleimide and more resistant to 45 °C treatment than is DNA synthesis in the transformed cell lines. N-Ethylmaleimide decreases the capacity of diploid cells to discriminate against BUdR, whereas heating increases it. Transformed cells treated with N-ethylmaleimide remain unable to discriminate against BUdR; some transformed lines, when heated at 45 °C, become less incapable of such discrimination.     

Conversion of dNTP to dNMP dependent on DNA synthesis in isolated Yoshida sarcoma nuclei

Nuclei isolated from Yoshida sarcoma cells had activity for conversion of dGTP to dGMP dependent on DNA synthesis. The ratio of nucleotide generation/generation + incorporation was 0.4 ± 0.1, indicating that approx. 40% of the incorporated dGMP was excised. Two lines of evidence indicated the dependence of this activity on DNA synthesis. (1) The activity was observed only in the presence of ATP, which is essential for nuclear DNA synthesis. (2) Inhibitors of DNA synthesis, such as N-ethylmaleimide, aphidicolin, spermine and KCl, also inhibited ATP- or DNA synthesis-dependent dGMP generation. Although nuclei contain nucleoside triphosphatase (N-nucleotidase), this enzyme was not involved appreciably in DNA synthesis-dependent dGMP generation. The reason for this was explained by the following findings. (a) Inhibitors did not decrease dGMP production in the complete absence of DNA synthesis. (b) Inhibitors did not inactivate N-nucleotidase to the same degree as they inhibited DNA synthesis-dependent dGMP generation. (c) Addition of ATP reduced dGTP hydrolysis catalyzed by N-nucleotidase. (d) GDP had no appreciable effect on DNA synthesis-dependent dGMP generation, but had a diluting effect on dGMP production catalyzed by N-nucleotidase. These results show that the pathway of dGMP generation in isolated nuclei was switched on addition of ATP from a N-nucleotidase-catalyzed one to a DNA polymerase-exonuclease-catalyzed one.     

Induction of 6-thioguanine resistance in synchronized human fibroblast cells treated with methyl methanesulfonate, N-acetoxy-2-acetylaminofluorene and N-ethyl-N′-nitro-N-nitrosoguanidine

Chemical induction of 6-thioguanine resistance was studied in synchronized human fibroblast cells. Cells initially grown in a medium lacking arginine and glutamine for 24 h ceased DNA synthesis and failed to enter the S phase. After introduction of complete medium, the cells progressed to the S phase after 16 h. DNA synthesis peaked 20 h after removal of nutrient stress and declined.Mutations were induced in S-phase cells by methyl methanesulfonate (MMS), N-acetoxy-2-acetylaminofluorene (NA-AAF) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Chemical treatments resulted in an increase in the absolute number of mutant colonies and in a dose-dependent mutation frequency. In this report, we show that NA-AAF evokes a temporal pattern of mutation in synchronized cells, with such mutations being induced only during the S phase. Evidence indicates that presence of S-phase cells in the treated cultures is a prerequisite for the induction of mutations.     

A nonalkaline method for isolating sequencing-ready plasmids

We describe a simple method of isolating plasmid DNA directly from Escherichia coli culture medium by addition of lithium acetate and Sodium dodecyl sulphate, followed by centrifugation and alcohol precipitation. The plasmid is sufficiently pure that it can be used in many enzyme-based reactions, including DNA sequencing and restriction analysis. Chromosomal DNA contamination is significantly reduced by pretreatment of the culture with DNase I, suggesting that much of the contaminant is associated with permeable dead cells. Chromosomal DNA contaminant can also be selectively denatured without damage to the supercoiled plasmid by alkaline denaturation in an arginine buffer or heat treatment in the presence of urea or N,N-dimethylformamide.     

DNA repair and chromosomal stability in the alkylating agent-hypersensitive Chinese hamster cell line 27-1

27-1 is a mutant of Chinese hamster ovary cells (CHO cells) that is hypersenstivie to the toxic effects of ultraviolet light, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and other monofunctional alkylating agents. We show here that the enhanced MNNG sensitivity of these cells is not due to alterations in the amount of DNA methylation products introduced nor by a defect in the first step of removal of the main alkylation products 7-methylguanine and 3-methyladenine. However, these mutant cells perform more DNA repair synthesis after treatment with MNNG than normal CHO-9 cells. This observation might indicate a possible defect of a ligase involved in sealing DNA repair patches.27-1 cells did not show elevated frequencies of sister-chromatid exchange and chromosomal aberration induced by MNNG. The data show that MNNG-induced cell killing is not necessarily related to increased chromosomal instability.     

Retinal horizontal cells lacking Rb1 sustain persistent DNA damage and survive as polyploid giant cells

The retinoblastoma tumor susceptibility gene, Rb1, is a key regulator of the cell cycle, and mutations in this gene have been found in many human cancers. Prior studies showed that retina-specific knockout of Rb1 in the mouse results in the formation of abnormally large horizontal cells, but the development, fate, and genomic status of these cells remain unknown. In this study, we conditionally inactivate Rb1 in early retinal progenitors and show that the loss of Rb1 leads to the rapid degeneration of most retinal cells except horizontal cells, which persist as giant cells with aberrant centrosome content, DNA damage, and polyploidy/aneuploidy. We observed inappropriate cell cycle entry of Rb1-deficient horizontal cells during the first postnatal weeks, which dropped off abruptly by P30. Despite extensive DNA damage in Rb1-deficient horizontal cells, these cells can still enter mitosis. Adult Rb1-deficient horizontal cells display elevated DNA content (5N–34N) that varied continuously, suggesting the presence of aneuploidy. We also found evidence of supernumerary and disoriented centrosomes in a rare population of mitotic cells in the mutant retinas. Overall our data demonstrate that horizontal cells are a remarkably robust cell type and can survive for months despite extensive DNA damage and elevated genome content.     

Detection of mutagen-induced lesions in isolated DNA by marker rescue of Bacillus subtilis phage phi 105

A new mutagenesis assay is described which detects the induction of forward mutations in isolated DNA. The assay utilizes replicative from DNA of the temperate Bacillus subtilis phage φ105 and tests the ability of chemicals to induce lesions which inactivate phage genes involved in lysogen formation. There is a cluster of such genes tightly linked to the φ105 genetic marker Jsus11 which restricts the host range of the phage to cells capable of suppressing sus mutations. In the actual assay chemically treated DNA, from wild-type J⁺ phage, is added to competent cells which are infected with φ105Jsus11. Wild-type phage, capable of producing plaques on cells which are nonpermissive for φ105Jsus11, are produced by recombination between the added chemically-treated DNA and infecting φ105Jsus11 DNA. If the added DNA also carried mutagenic lesions in any of the genes controlling lysogeny, clear plaque mutants are produced which are readily distinguishable from the turbid plaquing wild-type phage. This report demonstrates the capacity of this marker rescue-based assay to detect as mutagens the following DNA-reactive chemicals: hydroxylamine (HA); N-methyl-N′-nitro-N-nitrosoguanidine (MNNG); chloroacetaldehyde (CAA); propylene oxide (PO) and N-acetyl-N-acetoxy-2-amino-fluorene (AAAF). The effect of using a host cell, defective for excision repair, on the sensitivity with which the assay detected the mutagenic activities of CAA, PO and AAAF also was examined.The new mutagenesis assay offers 2 advantages over several other previously described transformation-based assays: (1) in contrast to assays based on the induction of ribosome-associated drug resistances, the new assay can detect frameshift as well as base-substitution-type mutagens and (2) the mutants generated can be detected at high plating densities. The assay thus may be useful for general mutagen screening especially with highly bactericidal compounds which are not readily tested in other microbial assays.     

Absence of DNA repair replication after chemical mutagen damage in Vicia faba

Exposure of human (Hela) cells to the mutagens 4-nitroquinoline-1-oxide (4NQO) and N-methyl-N′-nitro-nitrosoguanidine (MNNG) produces damage in DNA that is repaired by a mechanism involving the insertion of new bases into DNA (repair replication). Vicia faba root tips, either from soaked seeds containing non-proliferating cells or from growing roots, do not perform detectable amounts of repair replication even though the mutagens inhibit DNA synthesis and cause chromosome aberrations. In view of similar failures to resolve excision in Chlamydomonas, Haplopappus, and Nicotiana after irradiation with UV light and in Vicia faba after X-irradiation it appears that plants in general might lack this repair process.     

Involvement of DNA replication and repair in mutagenesis of Haemophilus influenzae induced by N-nitrosocarbaryl

A temperature-sensitive DNA synthesis mutant of Haemophilus influenzae (strain dna9) was treated with the N-nitroso compound N-nitrosocarbaryl, then incubated at the permissive (36°) and nonpermissive (41°) temperatures. At various times lysates were made and used to transform a second culture to novobiocin resistance (a measure of the extent of mutation fixation). At the permissive temperature mutation fixation continued approximately linearly during at least half of the first round of DNA replication after treatment with N-nitrosocarbaryl. In the absence of DNA replication (41°), most but not all of the mutation fixation was eliminated. The nonreplicative type of mutation fixation was greater after treatment with a higher concentration of N-nitrosocarbaryl. The data indicate that premutational lesions occur over the entire chromosome and that the bulk of the mutation fixation requires DNA replication, but that a process independent of replication, quite possibly an erro-prone repair system, also is responsible for part of the mutation fixation in cells exposed to alkylating agents.When strain dna9 was treated with N-nitrosocarbaryl and then incubated at 41° for some time (stopping DNA replication and the bulk of the mutation fixation) before being grown at 36°, a large decrease in the final frequency was seen. This suggests that a repair mechanism still functional in the absence of DNA replication is capable of removing premutational lesions from H. influenzae DNA.