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.     

Bleomycin-induced DNA repair synthesis in permeable human fibroblasts: mediation of long-patch and short-patch repair by distinct DNA polymerases

Treatment of permeable human fibroblasts with bleomycin elicits DNA repair synthesis that is only partially sensitive to aphidicolin, an inhibitor of mammalian DNA polymerases alpha and delta. Inhibition of long-patch repair synthesis by omission of the three unlabeled deoxyribonucleoside triphosphates (dNTPs) selectively eliminates the aphidicolin-sensitive component. The majority of this residual aphidicolin-resistant repair synthesis is contained in ligated patches as revealed by resistance to exonuclease III. Determination of repair patch length by bromodeoxyuridine-induced density shift under conditions where essentially all of the repair synthesis is sensitive or resistant to aphidicolin yielded values of approximately 20 and 4 nucleotides per patch, respectively. On the basis of these data and the relative sensitivity of bleomycin-induced repair synthesis to N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP), 2',3'-dideoxythymidine 5'-triphosphate (ddTTP), and N-ethylmaleimide (NEM), long-patch repair is attributed to DNA polymerase delta and short-patch repair to DNA polymerase beta.     

DNA polymerase requirements for parvovirus H-1 DNA replication in vitro.

An in vitro system using nuclei from parvovirus H-1-infected cells was used to characterize the influence of inhibitors of mammalian DNA polymerases on viral DNA synthesis. The experiments tested the effects of aphidicolin, which is highly specific for DNA polymerase alpha, and 2',3'-dideoxythymidine-5'-triphosphate (ddTTP), which inhibits cellular DNA polymerases in the order gamma greater than beta greater than alpha. Both aphidicolin and ddTTP were inhibitory, indicating that both polymerase alpha and a ddttp-sensitive enzyme are required for viral DNA synthesis. This was seen more clearly in kinetic measurements, which indicated an initial period of rapid DNA synthesis with the participation of polymerase alpha, followed by a period of less rapid, but more sustained, rate of DNA synthesis carried out by a ddTTP-sensitive enzyme, probably polymerase gamma. One interpretation of the results is that polymerase alpha functions in a strand displacement stage of the viral DNA replication mechanism, whereas polymerase gamma serves to convert the displaced single strands back to double-strand replicative form.     

The role of DNA polymerases alpha, beta and gamma in nuclear DNA synthesis.

The effects of the inhibitors 2'3' dideoxythymidine triphosphate (ddTTP) and 1-beta-D-arabinofuranosyl cytosine triphosphate (araCTP) on DNA synthesis in isolated S-phase HeLa S3 nuclei have been examined. These effects are compared with the effects of the same inhibitors in partially purified preparations of DNA polymerases alpha and beta. The effect of ddTTP on partially purified DNA polymerase gamma was also tested. DNA polymerases beta and gamma were very sensitive to ddTTP whereas DNA polymerase alpha and DNA synthesis in isolated nuclei were quite resistant. The synthesis and subsequent ligation of primary DNA pieces ('Okazaki fragments') were not affected by the presence of this inhibitor. DNA synthesis in isolated nuclei and DNA polymerase alpha activity were very sensitive to araCTP whereas DNA polymerase beta was almost totally resistant to the inhibitor. The results indicate a major role for DNA polymerase alpha in DNA replication.     

Dideoxynucleoside triphosphates inhibit a late stage of SV40 DNA replicationin vitro

Summary The role of DNA polymerases in the replication of SV40 DNA was studied using a T-antigen-dependent assay supplemented with a human KB cell extract. Inhibition of DNA polymerase α by addition of aphidicolin or monoclonal antibodies prevented DNA synthesis, confirming the requirement for this enzyme in replication. The replication process was unaffected by ddTTP at a concentration (5 μM) inhibitory to DNA polymerases β and γ, however, higher concentrations of ddTTP (200 μM) caused an apparent accumulation of relaxed circular plasmid with a concomitant decrease in DNA synthesis. An analysis of this replication intermediate indicated that it was formed during the replication reaction and that the replicative cycle was nearly complete. A kinetic study of ddTTP inhibition strongly suggested DNA polymerase ε (PCNA-independent DNA polymerase δ) was the target of the inhibitor and that this enzyme functions during the final stages of DNA replication.     

The roles of DNA polymerases alpha, beta, and gamma in DNA repair synthesis induced in hamster and human cells by different DNA damaging agents

The involvement of DNA polymerases alpha, beta, and gamma in DNA repair synthesis was investigated in subcellular preparations of cultured hamster and human cells. A variety of DNA damaging agents, including bleomycin, neocarzinostatin, UV irradiation, and alkylating agents, were utilized to induce DNA repair. The sensitivity of repair synthesis, as well as replicative synthesis and purified DNA polymerase beta activity, to inhibition by the DNA polymerase inhibitors dideoxythymidine triphosphate, aphidicolin, cytosine arabinoside triphosphate, and N-ethylmaleimide was determined. No evidence was obtained for a major role of polymerase gamma in any type of repair synthesis. In both hamster and human cells, the sensitivity of bleomycin- and neocarzinostatin-induced repair synthesis to ddTTP inhibition was essentially identical with that observed for purified polymerase beta, indicating these repair processes proceeded through a mechanism utilizing polymerase beta. Repair synthesis induced by UV irradiation and alkylating agents was not sensitive to ddTTP, indicating repair of these lesions occurred through a pathway primarily utilizing a different DNA polymerase; presumably polymerase alpha. However, replicative synthesis was much more sensitive to polymerase alpha inhibitors than was repair synthesis induced by UV irradiation or alkylating agents. Neither the amount of DNA damage nor the amount of induced repair synthesis influenced the degree to which the different DNA polymerases were involved in repair synthesis. The possibility that "patch size" or the actual type of DNA damage determines the extent to which different polymerases participate in DNA repair synthesis is discussed.     

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.     

DNA single stranded gaps formed during DNA repair synthesis induced by methyl methanesulfonate are filled by sequential action of aphidicolin- and dideoxythymidine sensitive DNA polymerases in HeLa cells

DNA repair synthesis induced by methyl methanesulfonate in preconditioned HeLa cells in which DNA replicative synthesis had been highly suppressed was inhibited by aphidicolin (an inhibitor of DNA polymerases and ) and dideoxythymidine (ddThR, an inhibitor of DNA polymerase ). Incomplete repair patches sensitive to exonuclease III were accumulated in the presence of aphidicolin while not in the presence of ddThR. These patches were comopleted by the combined action of Klenow fragment and T4 DNA ligase, indicating that the single-stranded gaps were formed during the repair synthesis. Moreover, ddThR had little effect on the repair synthesis in the presence of aphidicolin. Thus, the results suggest that the single-stranded gaps may be sealed first by aphidicolin-sensitive polymerase followed by ddThR-sensitive DNA polymerase on the same site of the repair patch.Abbreviations ddThR dideoxythymidine - MMS methyl methanesulfonate - dNTP deoxynucleoside triphosphate     

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.     

几种药物对小鼠腹水癌细胞中一种DNA多聚酶及酶与模板复合体的作用

在小鼠艾氏腹水癌细胞中存在着一种DNA多聚酶及其与DNA模板的复合体。以不同药物对部分纯化的酶蛋白和复合体进行抑制实验,发现Aphidicolin、溴化乙锭、新生霉素和肝素均不同程度地抑制复合体和酶蛋白的活性;但酶蛋白和复合体对双脱氧胸苷三磷酸(ddTTP)不敏感。另外还发现复合体较酶蛋白对抑制剂有较强的抗性。     

The mode of action of 1-beta-D-arabinofuranosylcytosine in inhibiting DNA repair; new evidence using a sensitive assay for repair DNA synthesis and ligation in permeable cells

Mammalian cells permeabilised by treatment with saponin are capable of UV excision repair. We have developed an assay system which permits measurement of the later stages of repair, i.e. repair synthesis and ligation. Incomplete repair sites are accumulated in UV-irradiated cells by incubating them with DNA synthesis inhibitors hydroxyurea and aphidicolin. On removal of the inhibitors at the time of permeabilisation, these incomplete sites, detected as DNA breaks, are rapidly ligated in a reaction that requires deoxyribonucleoside triphosphates and is blocked by aphidicolin. Thus ligation is possible only after a significant amount of DNA synthesis. We have used the assay to clarify the mode of inhibition of DNA repair by 1-beta-D-arabinofuranosylcytosine (ara C), another DNA polymerase inhibitor. It is well known that incomplete repair sites accumulated in whole cells with ara C are ligated at a slow rate, if at all. The hypothesis that ara C blocks or reduces further polymerisation after its incorporation into repair patches is disproved by our demonstration that, in permeable cells, the accumulated DNA breaks are ligated very rapidly. The likely explanation of the action of ara C is that, once phosphorylated, it remains in the cell as ara CTP and continues to inhibit polymerisation through competition with dCTP; in permeable cells, it readily leaks out.     

Induction of DNA ligase I by 1-beta-D-arabinosylcytosine and aphidicolin in MiaPaCa human pancreatic cancer cells

Exposure of MiaPaCa cells to 1-beta-D-arabinosylcytosine (ara-C) resulted in an increase in DNA ligase levels up to threefold compared to that in the untreated control cells, despite significant growth inhibition. Increased levels of DNA ligase I protein appear to correlate with the appearance of increased mRNA levels. The [(3)H]thymidine incorporation experiment and the biochemical assay of total polymerase activity revealed that an increase in DNA ligase I levels after treatment with ara-C was not accompanied by an increase of DNA synthesis or an increased presence of DNA polymerase activity inside cells. When cells resumed DNA synthesis after drug treatment, DNA ligase I levels began to drop, indicating that increased DNA ligase I is not required for DNA synthesis. An increase in DNA ligase I was also observed in cells treated with aphidicolin, another inhibitor of DNA synthesis that inhibits DNA polymerases without incorporating itself into DNA, indicating that an increase in DNA ligase I levels could be caused by the arrest of DNA replication by these agents. Interestingly, caffeine, which is a well-known inhibitor of DNA damage checkpoint kinases, abrogated the increase in DNA ligase I in MiaPaCa cells treated with ara-C and aphidicolin, suggesting that caffeine-sensitive kinases might be important mediators in the pathway leading to the increase in DNA ligase I levels in response to anticancer drugs, including ara-C and aphidicolin. We propose that ara-C and aphidicolin induce damage to the DNA strand by arresting DNA replication forks and subsequently increase DNA ligase I levels to facilitate repair of DNA damage.     

Excision repair in permeable arrested human skin fibroblasts damaged by UV (254 nm) radiation: evidence that alpha- and beta-polymerases act sequentially at the repolymerisation step

We have characterised far-ultraviolet-radiation-induced DNA-repair synthesis in permeabilised arrested (non-dividing) primary human skin fibroblasts. Approximately half the maximum repair synthesis is seen after a UV fluence of 4.0 Jm-2 and little additional incorporation was observed at fluences above 20.0 Jm-2. UV-damaged permeable cells were treated with specific inhibitors of DNA polymerase alpha and beta, both alone and in combination. The degree of inhibition of repair incorporation by aphidicolin indicates that polymerase alpha is involved in the majority (85-90%) of repair synthesis after both high and low (less than 4.0 Jm-2) UV fluences. Dideoxythymidine triphosphate seems able to inhibit DNA-repair synthesis only when polymerase alpha is fully or almost fully functional, indicating that polymerase beta is unable to substitute in repair for an alpha polymerase blocked by aphidicolin. These data suggest that the two enzymes may act sequentially to complete repair patches rather than acting independently.     

Studies of the identity of the unknown in protein hydrolysates

DNA synthesis in the adenovirus DNA replication complex, containing host DNA polymerases-α and -γ, was inhibited completely by aphidicolin and by 2′,3′-dideoxythymidine triphosphate (ddTTP). Double reciprocal plots of DNA polymerase activity in the replication complex against each dNTP gave a straight line although the complex contained two species of DNA polymerase. Inhibition by aphidicolin of DNA polymerase activity was competitive with dTTP but that of purified DNA polymerase-α isolated from adenovirus infected KB cells was competitive with dCTP. The above results suggest that DNA polymerases-α and -γ are integrated in the replication complex to behave as a single enzyme.     

Xeroderma pigmentosum variant (XP-V) correcting protein from HeLa cells has a thymine dimer bypass DNA polymerase activity.

Xeroderma pigmentosum variant (XP-V) represents one of the most common forms of this cancer-prone DNA repair syndrome. Unlike classical XP cells, XP-V cells are normal in nucleotide excision repair but defective in post-replication repair. The precise molecular defect in XP-V is currently unknown, but it appears to be a protein involved in translesion synthesis. Here we established a sensitive assay system using an SV40 origin-based plasmid to detect XP-V complementation activity. Using this system, we isolated a protein from HeLa cells capable of complementing the defects in XP-V cell extracts. The protein displays novel DNA polymerase activity which replicates cyclobutane pyrimidine dimer-containing DNA templates. The XPV polymerase activity was dependent on MgCl2, sensitive to NEM, moderately sensitive to KCl, resistant to both aphidicolin and ddTTP, and not stimulated by PCNA. In glycerol density gradients, the activity co-sedimented with a 54 kDa polypeptide at 3.5S, indicating that the monomeric form of this polypeptide was responsible for the activity. The protein factor corrected the translesion defects of extracts from three XPV cell strains. Bypass DNA synthesis by the XP-V polymerase occurred only in the presence of dATP, indicating that it can incorporate only dATP to bypass a di-thymine lesion.     

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.     

Inhibition by aphidicolin and dideoxythymidine triphosphate of a multienzyme complex of DNA synthesis from human cells

A multienzyme complex consisting of DNA polymerase and several DNA precursor-synthesizing enzymes was solubilized by gentle lysis of cultured human cells. This complex channelled the distal precursor [3H]dTMP into DNA. The patterns of inhibition of the complex by aphidicolin and dideoxythymidine triphosphate (ddTTP) suggested that the complex contained the replicative DNA polymerase, polymerase alpha. Inhibition by ddTTP was competitive with dTTP. This was exploited to estimate the effective concentration of [3H]dTTP at the site of DNA synthesis during channelling of [3H]dTMP into DNA. The estimated concentration (about 50 microM) was so high as to suggest that the solubilized complex was able to functionally compartmentalize DNA precursors.     

Studies on fertilization in the teleost IV. Effects of aphidicolin and camptothecin on chromosome formation in fertilized medaka eggs

To clarify the mechanisms of fish fertilization, the effects of inhibitors of DNA polymerase-alpha and DNA topoisomerases on nuclear behavior before and after fertilization were examined in eggs of the medaka, Oryzias latipes. Eggs underwent the fertilization process from sperm penetration to karyogamy of pronuclei, even when inseminated and incubated in the continuous presence of aphidicolin (DNA polymerase alpha inhibitor), camptothecin (DNA topoisomerase I inhibitor), etoposide, or beta-lapachone (DNA topoisomerase II inhibitor). However, continuous treatment with aphidicolin or camptothecin during fertilization inhibited the formation of sister chromosomes that were normally separated into blastomeres at the time of the subsequent cleavage. Sister chromosome formation appeared concomitantly with an increase in histone H1 kinase activity at the end of DNA synthesis, 30 min post insemination. However, non-activated eggs that were inseminated in saline containing anesthetic MS222 and aphidicolin had high levels of histone H1 kinase and MAP kinase activities, and transformation of the penetrated sperm nucleus to metaphase chromosomes occurred even in the presence of aphidicolin or camptothecin. The male chromosomes were normally separated into two anaphase chromosome masses upon egg activation. These results suggest that DNA polymerase alpha or DNA topoisomerase I, but not DNA topoisomerase II, may be required for the process by which the mitotic interphase nucleus transforms to separable metaphase chromosomes while the activity of MAP kinase is low, unlike the situation in meiotic division, during which MAP kinase activity is high and DNA replication is not required.     

Aphidicolin inhibits repair of DNA in UV-irradiated human fibroblasts

Aphidicolin, a specific inhibitor of DNA polymerase α, is shown to inhibit DNA repair in human diploid fibroblasts. Although aphidicolin has no apparent effect on the DNA of unirradiated cells, it causes a large number of strand breaks to accumulate in UV-irradiated cellular DNA. The number of breaks is the same as the number observed following a similar dose of ultraviolet light when cells are treated with arabinofuranosyl cytosine (araC) and hydroxyurea (HU), known inhibitors of repair. Moreover, two-dimensional paper chromatography shows that aphidicolin completely blocks removal of pyrimidine dimers. These observations are discussed in light of the proposed roles of DNA polymerases α β in DNA replication and repair and the action of aphidicolin on polymerase α.     

DNA replication and UV-induced DNA repair synthesis in human fibroblasts are much less sensitive than DNA polymerase alpha to inhibition by butylphenyl-deoxyguanosine triphosphate.

In mammalian cells, both semiconservative DNA replication and the DNA repair patch synthesis induced by high doses of ultraviolet radiation are known to be inhibited by aphidicolin, indicating the involvement in these processes of one or both of the aphidicolin-sensitive DNA polymerases, alpha and/or delta. In this paper, N2-(p-n-butylphenyl)-2'-deoxyguanosine-5'-triphosphate, a strong inhibitor of polymerase alpha and a weak inhibitor of polymerase delta, is used to further characterize the DNA polymerase(s) involved in these two forms of nuclear DNA synthesis. In permeable human fibroblasts, DNA replication and ultraviolet-induced DNA repair synthesis are more resistant to the inhibitor than DNA polymerase alpha by factors of approximately 500 and 3000, respectively. These findings are most consistent with the involvement of DNA polymerase delta in these processes.