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.     

Identification of a DNA polymerase beta-like form in Drosophila melanogaster adult flies.

DNA polymerase beta is widely distributed in the eukariotes. So far, few examples are known in which a DNA polymerase alpha -like form alone is reported. Surprisingly, DNA polymerase beta was not detected in Drosophila embryos, while it is present in the cells of multicellular species from sponge to mammals. In view of the relevance of Drosophila as a model biological system for studying the role of the various DNA metabolism enzymes in vivo we have reinvestigated the presence of the DNA polymerase beta-like form in Drosophila adult flies.Here we report the occurrence in Drosophila melanogaster adult flies of a DNA polymerase activity that, for its NEM(1) resistance, template specificity, sensitivity to ddTTP, sedimentation coefficient and nuclear localization can be classified as a beta-like form.     

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.     

Immunoaffinity purification and properties of Drosophila melanogaster DNA polymerase alpha-primase complex

Two hybrid cell lines (DM88-5E12 and DM88-4C9) secreting monoclonal antibodies against DNA polymerase alpha-primase complex from Drosophila melanogaster Kc cells were established by immunizing mice with the complex partially purified by a conventional method. The IgG subclasses of both antibodies were IgG1. Both antibodies immunoprecipitated the DNA polymerase alpha-primase complex from D. melanogaster Kc cells. The DNA-polymerizing activity was neutralized by 4C9 antibody, but not by 5E12 antibody. The DNA priming activity was not neutralized by either antibody. These antibodies did not cross-react to HeLa DNA polymerase alpha-primase complex. A rapid, two-step purification of DNA polymerase alpha-primase complex from D. melanogaster Kc cell was carried out by 5E12 antibody column chromatography followed by single-stranded DNA cellulose column chromatography. The immunoaffinity-purified enzyme had both DNA-polymerizing and DNA-priming activities with the specific activities of 50,000 and 2,000 units/mg, respectively. The effects of aphidicolin, NEM, ddTTP, BuPdGTP, and DMSO on the enzyme activity showed that the purified enzyme was DNA polymerase alpha, but not DNA polymerase beta, gamma, or delta. The purified enzyme consisted of polypeptides with apparent molecular weights of 180 (and 145, 140, 130 kDa), 72, 63, 51, and 49 kDa. The 5E12 antibody was shown to bind to all the high-molecular-weight polypeptides, 180, 145, 140, and 130 kDa, by immuno-Western blotting analysis.     

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.     

Isolation of the DNA polymerase alpha core enzyme from mouse cells

DNA polymerase alpha has been purified from mouse hybridoma cells approximately 30,000-fold using a combination of conventional and high performance liquid chromatography. In contrast to previous characterizations of mammalian DNA polymerase alpha, this enzyme has a single high molecular mass polypeptide (185 kDa) in tight association with a 68-kDa polypeptide and this structure appears to be the core DNA polymerase of the mouse cells. The biochemically purified enzyme, with a specific activity of approximately 200,000 units/mg protein, has an estimated molecular mass by gel filtration chromatography of 240 kDa and sedimentation value of 9 S, consistent with the enzyme being a heterodimer of 185 and 68 kDa. The enzyme is sensitive to both N-ethylmaleimide and aphidicolin and insensitive to ddTTP. Using an activated DNA template, the apparent Km values for the deoxynucleotide triphosphates are approximately 0.5-1 microM. The purified DNA polymerase has neither exonuclease nor primase activities and is the predominant DNA polymerase alpha activity in the mouse cells.     

The DNA polymerases associated with the adenovirus type 2 replication complex: effect of 2''-3''-dideoxythymidine-5''-triphosphate on viral DNA synthesis.

An adenovirus (Ad) DNA replication complex extracted from infected HeLa nuclei could be purified free of the bulk of intracellular DNA polymerase activity by sedimetation in neutral sucrose gradients. However, the replication complex still retained some alpha and gamma DNA-polymerase activity. Since this complex is inhibited by 2', 3' dideoxythymidine-5'-triphosphate (ddTTP), an inhibitor of DNA polymerase gamma, a functional role for this enzyme in Ad DNA replication is suggested. Similar inhibition by ddTTP in intact Ad infected nuclei and comparable inhibition of Ad DNA synthesis in whole cells by dideoxythymidine (ddThy) are consistent with a role for DNA polymerase gamma. Uninfected HeLa nuclei or whole cells are not similarly inhibited by ddTTP or DDThy respectively. Such data does not rule out an additional functional role for other DNA polymerases, and recent experiments from this laboratory (1) suggest that DNA polymerase alpha is also involved in Ad DNA synthesis.     

A new DNA polymerase species from Drosophila melanogaster: a probable mus308 gene product.

Harris et al. [P.V. Harris, O.M. Mazina, E.A. Leonhardt, R.B. Case, J.B. Boyd, K.C. Burtis, Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes, Mol. Cell. Biol., 16 (1996) 5764-5771.] reported the molecular cloning of Drosophila mus308 gene, and its nucleotide and protein sequences similar to DNA polymerase I. In the present study, we attempted to find and isolate the gene product by purifying a DNA polymerase fraction not present in mus308 flies. A new DNA polymerase with properties different from those of any known polymerase species was identified and partially purified from the wild-type fly embryos through ten column chromatographies. The enzyme was resistant to aphidicolin, but sensitive to ddTTP and NEM. Human proliferating cell nuclear antigen (PCNA) and Drosophila replication protein A (RP-A) did not affect the polymerase activity. It preferred poly(dA)/oligo(dT) as a template-primer. The molecular mass was about 230 kDa with a broad peak region of 200 to 300 kDa in HiPrep16/30 Sephacryl S-300 gel filtration. These properties a different from those of all reported Drosophila polymerase classes such as alpha, beta, gamma, delta, epsilon and zeta and closely resemble those of the gene product expected from the nucleotide sequence. The new polymerase species appears to have ATPase and 3'-5' exonuclease activities as shown by the chromatographies.     

2',3'-Dideoxythymidine 5'-triphosphate inhibition of DNA replication and ultraviolet-induced DNA repair synthesis in human cells: evidence for involvement of DNA polymerase delta

It is well established that DNA replication and ultraviolet-induced DNA repair synthesis in mammalian cells are aphidicolin-sensitive and thus are mediated by one or both of the aphidicolin-sensitive DNA polymerases, alpha and/or delta. Recently, it has been shown that DNA polymerase delta is much more sensitive to inhibition by the nucleotide analogue 2',3'-dideoxythymidine 5'-triphosphate (ddTTP) than DNA polymerase alpha but is less sensitive than DNA polymerase beta [Wahl, A. F., Crute, J. J., Sabatino, R. D., Bodner, J. B., Marraccino, R. L., Harwell, L. W., Lord, E. M., & Bambara, R. A. (1986) Biochemistry 25, 7821-7827]. We find that DNA replication and ultraviolet-induced DNA repair synthesis in permeable human fibroblasts are also more sensitive to inhibition by ddTTP than polymerase alpha and less sensitive than polymerase beta. The Ki for ddTTP of replication is about 40 microM and that of repair synthesis is about 25 microM. These are both much less than the Ki of polymerase alpha (which is greater than 200 microM) but greater than the Ki of polymerase beta (which is less than 2 microM). These data suggest that DNA polymerase delta participates in DNA replication and ultraviolet-induced DNA repair synthesis in human cells.     

Effect of 2'',3''-dideoxythymidine-5''-triphosphate on HeLa cell in vitro DNA synthesis: evidence that DNA polymerase alpha is the only polymerase required for cellular DNA replication.

We have studied the effects of the nucleotide analogue, 2',3'-dideoxythymidine-5'-triphosphate (ddTTP) on replicative DNA synthesis in HeLa cell lysates. As previously demonstrated (1), such lysates carry out extensive DNA synthesis in vitro, at rates and in a fashion similar to in vivo DNA replication. We report here that all aspects of DNA synthesis in such lysates (total dNTP incorporation, elongation of continuous nascent strands, and the initiation, elongation, and joining of Okazaki pieces) are only slightly inhibited by concentrations of ddTTP as high as 100-500 micrometer when the dTTP concentration is maintained at 10 micrometer. This finding is consistent with the report by Edenberg, Anderson, and DePamphilis (2) that all aspects of replicative in vitro simian virus 40 DNA synthesis are also resistant to ddTTP. We also find, in agreement with Edenberg, Anderson, and DePamphilis (2), that DNA synthesis catalyzed by DNA polymerases beta or gamma is easily inhibited by ddTTP, while synthesis catalyzed by DNA polymerase alpha is very resistant. These observations suggest that DNA polymerase alpha may be the only DNA polymerase required for all aspects of cellular DNA synthesis.     

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.     

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.     

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 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.     

Mechanisms of inhibitions of DNA polymerase gamma by nucleotide analogues having anti-HIV activities.

Inhibition mechanisms of 5'-triphosphates of 3'-azido-3'-deoxythymidine (AZT-TP) and 3'-deoxythymidine (ddTTP) on extensively purified DNA polymerase gamma from bovine testes were examined by analysis of the products synthesized on singly primed M13mp18 single-stranded DNA or synthetic oligonucleotide template-primer in the presence of analogues. The results indicate that AZT-TP inhibits DNA polymerase gamma in competition with dTTP but is not incorporated into DNA, whereas ddTTP is incorporated into DNA and causes chain termination.     

Novikoff hepatoma deoxyribonucleic acid polymerase. Sensitivity of the beta-polymerase to sulfhydryl blocking agents.

Unlike other beta-class eukaryotic DNA polymerases, the enzyme purified from the Novikoff hepatoma is inhibited by both sulfhydryl blocking agents N-ethylmaleimide (NEM) and p-hydroxymercuribenzoate (pHMB). The degree of sensitivity varies depending on the enzyme purity, pH of the reaction, and the presence of sulfhydryl reducing agents. Novikoff beta-polymerase activity is unaffected by the presence of 2-mercaptoethanol (2-Me) or dithiothreitol (DTT); however, the combination of 2-mercaptoethanol and NEM or pHMB acts to reverse the inhibition of the sulfhydryl blocking agent. The reversal of inhibition involves more than just a titration of NEM with 2-mercaptoethanol since a) the combination of these two reagents actually stimulates the DNA polymerase, and b) dithiothreitol did not reverse the inhibition. Binding of the polymerase to DNA did not affect the enzyme sensitivity to NEM.     

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.     

Contribution of DNA polymerase delta to DNA replication in permeable CHO cells synchronized in S phase.

To evaluate the relative contributions of DNA polymerase alpha and DNA polymerase delta in chromosome replication during the S phase of the cell cycle, we have used the permeable cell system for replication as a functional assay. We carried out the analysis of DNA polymerases both in quiescent cells stimulated to proliferate and progress through the cell cycle (monolayers) and in actively growing cells separated into progressive stages of the cell cycle by centrifugal elutriation (suspension cultures). DNA polymerase alpha was measured by using the inhibitor butylphenyl dGTP at low concentrations. Using several inhibitors such as aphidicolin, ddTTP and butylphenyl dGTP, we found that DNA polymerase alpha and delta activity were coordinately increased during S phase and declined at the end. However, DNA polymerase delta was performing about 80% of the total replication and DNA polymerase alpha performed only 20%. This high ratio of DNA polymerase delta to DNA polymerase alpha replication activity was maintained throughout S phase in two entirely different experimental approaches.     

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.