The inhibition of DNA repair in primary rat hepatocyte cultures by aphidicolin: Evidence for the involvement of alpha polymerase in the repair process

The effect of aphidicolin on the repair of chemically induced DNA damage in rat hepatocytes was examined. Alkaline elution analysis of DNA damage and autoradiographic examination of unscheduled DNA synthesis both indicate that the repair of DNA damage was inhibited by aphidicolin. Because aphidicolin has been shown to be a specific inhibitor of alpha polymerase, these results suggest that the alpha polymerase plays an active role in the repair of rat hepatocyte DNA.     

Excision repair of DNA in the presence of aphidicolin

During excision repair of UV light or dimethyl sulphate (DMS)-induced damage to DNA the patch size for actively replicating KB or T98G cells is around 20 nucleotides. When confluent T98G cells or 'quiescent' KB cells are used the patch size is around 10 nucleotides. This value can be increased to around 20 nucleotides in T98G cells if a large excess of BrdUrd is included in the repair incubation medium. With 'quiescent' KB cells the patch size is not increased by excess BrdUrd. For all of these experimental conditions, when excision repair of UV or DMS damage takes place in the presence of aphidicolin, the patch size is found to be several times that found in its absence. Given the inhibitory specificity of aphidicolin for DNA polymerase alpha these results provide additional evidence that DNA polymerase alpha plays a role in the excision repair of DNA damaged by UV light or DMS. It is postulated that aphidicolin interrupts the processivity of the DNA polymerase alpha holoenzyme and allows an exonuclease to enlarge the repair site.     

Effect of DNA polymerase inhibitors on DNA repair in intact and permeable human fibroblasts: evidence that DNA polymerases delta and beta are involved in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine

The involvement of DNA polymerases alpha, beta, and delta in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated in human fibroblasts (HF). The effects of anti-(DNA polymerase alpha) monoclonal antibody, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), dideoxythymidine triphosphate (ddTTP), and aphidicolin on MNNG-induced DNA repair synthesis were investigated to dissect the roles of the different DNA polymerases. A subcellular system (permeable cells), in which DNA repair synthesis and DNA replication were differentiated by CsCl gradient centrifugation of BrdUMP density-labeled DNA, was used to examine the effects of the polymerase inhibitors. Another approach investigated the effects of several of these inhibitors on MNNG-induced DNA repair synthesis in intact cells by measuring the amount of [3H]thymidine incorporated into repaired DNA as determined by autoradiography and quantitation with an automated video image analysis system. In permeable cells, MNNG-induced DNA repair synthesis was inhibited 56% by 50 micrograms of aphidicolin/mL, 6% by 10 microM BuPdGTP, 13% by anti-(DNA polymerase alpha) monoclonal antibodies, and 29% by ddTTP. In intact cells, MNNG-induced DNA repair synthesis was inhibited 57% by 50 micrograms of aphidicolin/mL and was not significantly inhibited by microinjecting anti-(DNA polymerase alpha) antibodies into HF nuclei. These results indicate that both DNA polymerases delta and beta are involved in repairing DNA damage caused by MNNG.     

Excision repair in u.v. (254 nm) damaged non-dividing human skin fibroblasts: a major biological role for DNA polymerase alpha

We have used the eukaryotic DNA polymerase alpha inhibitor, aphidicolin, and the polymerase beta inhibitor, dideoxythymidine, to examine the role of these enzymes in excision repair of ultraviolet (u.v., 254 nm) damage induced in non-dividing (arrested) human skin fibroblasts. The effects of these drugs on u.v.-treated cells have been monitored using a simple and reproducible repair synthesis assay in parallel with viability measurements to determine the degree of inhibition of repair of potentially lethal damage. In agreement with previous studies using density gradients, repair synthesis induced by low fluences of u.v. (less than 3 J m-2) is relatively insensitive to inhibition by aphidicolin compared to high fluences where approximately 85 per cent inhibition is observed at the highest (20 micrograms/ml) aphidicolin concentration employed. However, repair of potentially lethal damage is inhibited by at least 90 per cent over the entire fluence range. Although dideoxythymidine led to considerable inhibition of repair synthesis, the result is probably an artifact under these in vivo conditions. The polymerase beta inhibitor was not toxic to u.v.-treated cells nor did it add to the toxicity of aphidicolin when the drugs were used in combination. We conclude that if the beta polymerase is involved in excision repair then its temporary (4 h) inhibition by dideoxythymidine is entirely reversible. In contrast, polymerase alpha appears to be an enzyme essential to the majority of biologically effective excision repair over the entire u.v. fluence range tested.     

Effects of aphidicolin and/or 2',3'-dideoxythymidine on DNA repair induced in HeLa cells by four types of DNA-damaging agents

The alkaline sucrose density gradient centrifugation method was modified to permit detection of 1 lesion/10(9) daltons of DNA. With this technique, the involvements of DNA polymerases in DNA repair of damage by dimethyl sulfate, UV irradiation, neocarzinostatin, and bleomycin were studied in HeLa cells with the aid of the DNA polymerase inhibitors aphidicolin and 2',3'-dideoxythymidine. DNA repair after UV-induced damage seemed to involve only polymerase alpha, while repair of damage by the other three agents involved both polymerase alpha and a non-alpha polymerase, probably polymerase beta. But repair after damage by dimethyl sulfate differed from that after damage by neocarzinostatin or bleomycin with respect to the co-operations of polymerase alpha and polymerase beta: in repair of dimethyl sulfate-induced damage, both polymerases operated on the same lesions, whereas after damage by neocarzinostatin or bleomycin, polymerase alpha and polymerase beta functioned independently on different lesions.     

Reduced repair of X-ray-induced DNA lesions in cells without functioning DNA polymerase alpha

X irradiation of cells induces damage in the DNA, which can be detected as fragmentation of the DNA in alkali. To examine whether DNA polymerase alpha plays a role in the X-ray-induced fragmentation of the DNA, cells with and without functioning DNA polymerase alpha have been compared. We have used the drug aphidicolin, which is a specific inhibitor of polymerase alpha. The results show that DNA of aphidicolin-treated cells is more easily fragmented in alkali than DNA of untreated cells. This is paralleled by a lower repair replication in cells without functioning DNA polymerase alpha. Hence polymerase alpha is involved in the repair process of lesions induced by X irradiation.     

DNA lesions in hyperthermic cell killing: effects of thermotolerance, procaine, and erythritol

When HeLa S3 cells were subjected to 45 degrees C hyperthermia, DNA lesions were detected by the use of the alkaline unwinding/hydroxylapatite method. The number of lesions formed was not affected when the cells were made thermotolerant by either an acute (15 min 44 degrees C + 5 h 37 degrees C) or a chronic (5 h 42 degrees C) pretreatment before 45 degrees C hyperthermia. The presence of 10 mM procaine (heat sensitizer) or 0.5 M erythritol (heat protector) during hyperthermia also had no effect on the rate of formation of heat-induced alkali labile DNA lesions. These observations do not support a concept where DNA lesions are considered to be the ultimate cause of hyperthermic cell killing. Both drugs, however, influenced the rate of repair of radiation-induced strand breaks when present during preirradiation heat treatment. We conclude that the initial number of heat-induced alkali labile DNA lesions is not directly related to cell survival. It cannot be excluded, however, that differences in posthyperthermic repair of these lesions may lead to a positive correlation between residual DNA damage and survival after the different experimental conditions.     

Characterization of DNA polymerase alpha activity from a mouse DNA temperature-sensitive mutant, strain tsFT20, which shows a defect in DNA polymerase alpha activity at restrictive temperatures

tsFT20 cells derived from mouse FM3A cells are DNA temperature-sensitive mutants, which have heat-labile DNA polymerase alpha activity. When tsFT20 cells were incubated at restrictive temperatures, intracellular levels of DNA polymerase alpha activity changed biphasically, showing an initial fast decrease (phase I) and a subsequent slow decrease (phase II). The activity of DNA polymerase alpha from tsFT20 cells cultured at a permissive temperature (33 degrees C) was greatly increased by the addition of glycerol or ethylene glycol to the reaction mixture, while little increase in enzyme activity was observed at any concentration of glycerol or ethylene glycol tested with the enzyme from the cells cultured at a restrictive temperature (39 degrees C) for 8 h (phase II). The activity of DNA polymerase alpha from wild-type cells was also increased by the addition of glycerol but the increase was much less than that in the tsFT20 cells. An in vitro preincubation experiment showed that DNA polymerase alpha from tsFT20 cells cultured at 33 degrees C very rapidly lost its ability to be stimulated by glycerol. Furthermore, the experiment using the extracts prepared from tsFT20 cells cultured at 39 degrees C for various periods showed that the ability to be stimulated by glycerol decreased with the duration of incubation time at 39 degrees C. DNA polymerase alpha from the revertants, which can grow at 39 degrees C and exhibit a partial recovery in heat stability of DNA polymerase alpha activity, showed an intermediate response to glycerol, between those of DNA polymerase alpha from tsFT20 and from the wild-type cells. Finally, it was observed that the level of enzyme activity that can be stimulated by glycerol correlated well with the DNA synthesizing ability of tsFT20 cells.     

Effect of nalidixic acid on DNA repair in rat hepatocytes

Nalidixic acid, a DNA topoisomerase inhibitor, has been reported to inhibit DNA repair in some mammalian systems. To investigate the effect of nalidixic acid on DNA repair in cultured rat hepatocytes, DNA damage was induced by ultraviolet light or N-methyl-N-nitro-N-nitrosoguanidine. The presence of aphidicolin, a DNA polymerase alpha inhibitor resulted in a decrease in DNA repair. Nalidixic acid had no inhibitory effect. Neither aphidicolin nor nalidixic acid induced DNA repair. These results indicate that nalidixic acid does not damage DNA or inhibit DNA repair processes in hepatocytes.     

Involvement of DNA polymerase alpha in host cell reactivation of UV-irradiated herpes simplex virus.

Aphidicolin is a potent inhibitor of both host cell DNA polymerase alpha and herpes simplex virus (HSV)-induced DNA polymerase but has no effect on DNA polymerases beta and gamma of host cells. By using an aphidicolin-resistant mutant (Aphr) of HSV, a possible involvement of DNA polymerase alpha in host cell reactivation of UV-damaged HSV was studied. Plaque formation by UV-irradiated Aphr was markedly inhibited by 1 microgram of aphidicolin per ml, which did not affect the plating efficiency of nonirradiated Aphr. Aphidicolin added before 12 h postinfection inhibited plaque formation by irradiated Aphr, which became aphidicolin insensitive after 36 h postinfection. The results strongly suggest that host cell DNA polymerase alpha is involved in the repair of UV-irradiated HSV DNA.     

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.     

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.     

Hyperthermic potentiation of unrejoined DNA strand breaks following irradiation

Previous reports have suggested that the potentiation of cellular radiation sensitivity by hyperthermia may be due to its inhibition of the repair of single-strand breaks in DNA. Such inhibition could result in increased numbers of unrejoined breaks at long times following irradiation, lesions that are presumed to be lethal to the cell. As a test of this hypothesis, the amounts of residual strand-break damage in cells following combined hyperthermia and ionizing radiation were measured. The results show that hyperthermia does significantly enhance the relative number of unrejoined strand breaks as measured by the technique of alkaline elution and that the degree of enhancement is dependent on both the temperature and duration of the hyperthermia treatment. For example, compared to unheated cells, the proportion of unrejoined breaks measured 8 hr after irradiation was increased by a factor of 1.5 in cells that were treated for 30 min at 43 degrees C, by a factor of 6 for cells treated for 30 min at 45 degrees C, and by a factor of 4 for cells treated at 43 degrees C for 2 hr. In experiments in which the sequence of heat and irradiation were varied, a high degree of correlation was observed between the resulting level of cell killing and the relative numbers of unrejoined strand breaks. The greatest effects on both of these parameters were observed in those protocols in which the irradiation was delivered either during, just before, or just after the heat treatment.     

Differential effect of aphidicolin on adenovirus DNA synthesis and cellular DNA synthesis.

There is strong evidence for a participation of DNA polymerase gamma in the replication of adenovirus (Ad) DNA. To study a possible additional role of DNA polymerase alpha we measured the effect of aphidicolin on viral DNA replication. In intact cells, aphidicolin inhibits Ad DNA synthesis weakly. The drug concentration required for 50% inhibition of Ad DNA replication was 300-400 fold higher than for a similar effect on cellular DNA synthesis. Such a differential inhibition was also observed in AGMK cells doubly infected with SV40 and the simian adenovirus SA7. No evidence was found for modification of aphidicolin in infected cells or for a change in aphidicolin sensitivity of DNA polymerase alpha after infection. The extent of inhibition of purified DNA polymerase alpha was dependent upon the dCTP concentration. The same situation was observed when DNA synthesis was studied in isolated nuclei from uninfected cells. However, in nuclei from Ad infected cells no effect of dCTP on aphidicolin sensitivity was found. These results were taken as evidence that DNA polymerase alpha does not participate in the replication of adenovirus DNA.     

Two different mechanisms are involved for the bleomycin-induced DNA repair synthesis in permeabilized HeLa cells

Bleomycin-induced DNA repair synthesis in the permeabilized HeLa cells was sensitive to aphidicolin, an inhibitor of DNA polymerase alpha and delta, and to dideoxythymidine triphosphate (ddTTP), a specific inhibitor of DNA polymerase beta. Upon combined treatment with these inhibitors, the DNA repair synthesis was inhibited to an even higher degree. This indicated that the aphidicolin- and ddTTP-sensitive DNA repair syntheses may occur by independent mechanisms. The structure of incomplete repair patches being accumulated in the presence of these inhibitors was investigated by digestion of DNA with exonuclease III after incubation with Klenow fragment and T4 DNA ligase. The results have suggested that the patch accumulating in the presence of aphidicolin is a single-stranded gap made by excision enzyme(s), whereas that accumulating in the presence of ddTTP may be generated by strand displacement.     

Repair of benzo[a]pyrene-initiated DNA damage in human cells requires activation of DNA polymerase alpha

Normal human fibroblasts treated with r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) yielded DNA polymerase alpha with elevated levels of activity, incorporated [3H]thymidine as a function of unscheduled DNA synthesis, and exhibited restoration of normal DNA-strand length as a function of unscheduled DNA synthesis. Lipoprotein-deficient fibroblasts treated with BPDE did not show elevated levels of DNA polymerase alpha activity, exhibited minimal [3H]thymidine incorporation, and had fragmented DNA after 24 h of repair in the absence of lipoprotein or phosphatidylinositol supplementation. When DNA polymerase beta activity was inhibited, cells with normal lipoprotein uptake exhibited [3H]thymidine incorporation into BPDE-damaged DNA but did not show an increase in DNA-strand length. DNA polymerase alpha activity and [3H]thymidine incorporation in lipoprotein-deficient fibroblasts increased to normal levels when the cells were permeabilized and low-density lipoproteins or phosphatidylinositol were introduced into the cells. DNA polymerase alpha isolated from normal human fibroblasts, but not from lipoprotein-deficient fibroblasts, showed increased specific activity after the cells were treated with BPDE. When BPDE-treated lipoprotein-deficient fibroblasts were permeabilized and 32P-ATP was introduced into the cells along with lipoproteins, 32P-labeled DNA polymerase alpha with significantly increased specific activity was isolated from the cells. These data suggest that treatment of human fibroblasts with BPDE initiates unscheduled DNA synthesis, as a function of DNA excision repair, which is correlated with increased activity of DNA polymerase alpha, and that increased DNA polymerase alpha activity may be correlated with phosphorylation of the enzyme in a reaction that is stimulated by low-density lipoprotein or by the lipoprotein component, phosphatidylinositol.     

Effects of gamma radiation and hyperthermia on DNA repair synthesis and the level of NAD+ in cultured human mononuclear leukocytes

DNA repair has been investigated, estimated by unscheduled DNA synthesis (UDS) and the cellular NAD+ pool, after exposing human mononuclear leukocytes to hyperthermia and gamma radiation separately and in combination. It was found that gamma radiation induced a decline in UDS with increasing temperature through the temperature region studied (37-45 degrees C). At 42.5 degrees C the gamma-ray-induced UDS was reduced to about 70% of that at 37 degrees C. Following gamma-ray damage the NAD+ pool dropped to about 20% of control values. Without hyperthermic treatment the cells completely recovered to the original level within 5 hr. Moderate hyperthermia (42.5 degrees C for 45 min) followed by gamma-ray damage altered the kinetics so that even after 8 hr the NAD+ pool had recovered to only 70% of the original level. After heat treatment at 44 degrees C for 45 min prior to gamma radiation the cells did not recover at all, presumably because of the cytotoxic effects from the combined treatment.     

Induced repair of DNA double-strand breaks at the G1/S-phase border

Exposure of human cells to ionizing radiation at the G1/S-phase border of the cell cycle leads to the production of repair patches of 3 nucleotides, representing the constitutive repair response, and very long repair patches (VLRP) of at least 150 nucleotides, representing an induced response. We examined the type of DNA damage that may signal this induced repair response using two chemicals that produce subsets of the damage induced by ionizing radiation. Treatment of cells at the G1/S-phase border with bleomycin, which produces a high proportion of DNA double-strand breaks, also leads to the production of VLRP of at least 130 nucleotides. In contrast, when cells were treated with hydrogen peroxide, which produces base modifications and single-strand breaks, no VLRP were observed. Thus it would appear that DNA double-strand breaks are the signal that leads to the induction of the VLRP. We also examined the relationship between the induced repair response and DNA replication. When cells are treated with hydroxyurea, under conditions that inhibit more than 98% of the DNA synthesis, prior to exposure to 5 Gy, repair patches of 3 and 150 nucleotides are found. This indicates that the longer repair patches are not a result of aberrant DNA replication. However, when cells are treated with the DNA polymerase inhibitor aphidicolin in combination with hydroxyurea and cytosine arabinoside, no induced long patches are found. These results indicate that DNA polymerase alpha, delta or epsilon is required for the synthesis of the VLRP.     

DNA polymerase delta mediates excision repair in growing cells damaged with ultraviolet radiation

In confluent, stationary phase cells, an aphidicolin-sensitive DNA polymerase mediates UV-induced excision repair, but the situation in growing cells is still controversial. The sensitivity of repair synthesis to aphidicolin, an inhibitor of DNA polymerases alpha and delta, was determined in growth phase and confluent normal human fibroblasts (AG1518) using several techniques. Repair synthesis in confluent cells was always inhibited by aphidicolin, no matter which measurement technique was used. However, the inhibition of repair synthesis in growth-phase cells by aphidicolin was only detectable when techniques unaffected by changes in nucleotide metabolism were used. We conclude that UV-induced repair synthesis in growing cells is actually aphidicolin sensitive, but that this inhibition can be obscured by changes in nucleotide metabolism. Employing butylphenyl-deoxyguanosine triphosphate, a potent inhibitor of polymerase alpha and a weak inhibitor of delta, we have obtained evidence that polymerase delta is responsible for repair synthesis in growth-phase cells following UV irradiation.     

Effect of specific enzyme inhibitors on replication, total genome DNA repair and on gene-specific DNA repair after UV irradiation in CHO cells.

We have studied the effect of some specific enzyme inhibitors on DNA repair and replication after UV damage in Chinese hamster ovary cells. The DNA repair was studied at the level of the average, overall genome and also in the active dihydrofolate reductase gene. Replication was measured in the overall genome. We tested inhibitors of DNA polymerase alpha and delta (aphidicolin), of poly(ADPr) polymerase (3-aminobenzamide), of ribonucleotide reductase (hydroxyurea), of topoisomerase I (camptothecin), and of topoisomerase II (merbarone, VP-16). In addition, we tested the effect of the potential topoisomerase I activator, beta-lapachone. All of these compounds inhibited genome replication and all topoisomerase inhibitors affected the overall genome repair; beta-lapachone stimulated it. None of these compounds had any effect on the gene-specific repair.