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.     

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.     

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.     

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.     

DNA repair synthesis in mouse spermatogenesis involves DNA polymerase beta activity

The role of DNA polymerase alpha-DNA primase complex and DNA polymerase beta in DNA replication and ultraviolet-induced DNA repair synthesis has been analyzed in mouse spermatogenesis. Autoradiographic experiments with germ cells in culture, indicating an involvement of DNA polymerase alpha and/or delta in DNA replication, and of DNA polymerase beta in DNA repair synthesis, have been confirmed by studying partially purified enzymes. These findings support the idea that, different from other biological systems, in meiotic and post meiotic male mouse germ cells DNA polymerase beta is the main DNA polymerase form needed for DNA repair.     

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 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.     

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.     

In situ enzymology of DNA replication and ultraviolet-induced DNA repair synthesis in permeable human cells

Using permeable diploid human fibroblasts, we have studied the deoxyribonucleoside triphosphate concentration dependences of ultraviolet- (UV-) induced DNA repair synthesis and semiconservative DNA replication. In both cell types (AG1518 and IMR-90) examined, the apparent Km values for dCTP, dGTP, and dTTP for DNA replication were between 1.2 and 2.9 microM. For UV-induced DNA repair synthesis, the apparent Km values were substantially lower, ranging from 0.11 to 0.44 microM for AG1518 cells and from 0.06 to 0.24 microM for IMR-90 cells. Control experiments established that these values were not significantly influenced by nucleotide degradation during the permeable cell incubations or by the presence of residual endogenous nucleotides within the permeable cells. Recent data implicate DNA polymerase delta in UV-induced repair synthesis and suggest that DNA polymerases alpha and delta are both involved in semiconservative replication. We measured Km values for dGTP and dTTP for polymerases alpha and delta, for comparison with the values for replication and repair synthesis. Km values for polymerase alpha were 2.0 microM for dGTP and 5.0 microM for dTTP. For polymerase delta, the Km values were 2.0 microM for dGTP and 3.5 microM for dTTP. The deoxyribonucleotide Km values for DNA polymerase delta are much greater than the Km values for UV-induced repair synthesis, suggesting that when polymerase delta functions in DNA repair, its characteristics are altered substantially either by association with accessory proteins or by direct posttranslational modification. In contrast, the deoxyribonucleotide binding characteristics of the DNA replication machinery differ little from those of the isolated DNA polymerases.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

DNA repair synthesis in human fibroblasts requires DNA polymerase delta

When UV-irradiated cultured diploid human fibroblasts were permeabilized with Brij-58 then separated from soluble material by centrifugation, conservative DNA repair synthesis could be restored by a soluble factor obtained from the supernatant of similarly treated HeLa cells. Extensive purification of this factor yielded a 10.2 S, 220,000-dalton polypeptide with the DNA polymerase and 3'- to 5'-exonuclease activities reported for DNA polymerase delta II (Crute, J. J., Wahl, A. F., and Bambara, R. A. (1986) Biochemistry 25, 26-36). Monoclonal antibody to KB cell DNA polymerase alpha, while binding to HeLa DNA polymerase alpha, did not bind to the HeLa DNA polymerase delta. Moreover, at micromolar concentrations N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) were potent inhibitors of DNA polymerase alpha, but did not inhibit the DNA polymerase delta. Neither purified DNA polymerase alpha nor beta could promote repair DNA synthesis in the permeabilized cells. Furthermore, under conditions which inhibited purified DNA polymerase alpha by greater than 90%, neither monoclonal antibodies to DNA polymerase alpha, BuPdGTP, nor BuAdATP was able to inhibit significantly the DNA repair synthesis mediated by the DNA polymerase delta. Thus, it appears that a major portion of DNA repair synthesis induced by UV irradiation might be catalyzed by DNA polymerase delta. When xeroderma pigmentosum human diploid fibroblasts were utilized, DNA repair synthesis dependent upon ultraviolet light could be restored by addition of both T4 endonuclease V and DNA polymerase delta, but not by addition of either one alone. This result suggests that cytosol-depleted permeabilized DNA repair-defective human fibroblasts and HeLa DNA polymerase delta might be exploited to provide a functional assay for purifying active DNA repair factors from DNA repair-proficient cells without a preknowledge of their function.     

Effects of 2',3'-dideoxythymidine triphosphate on replicative DNA synthesis and unscheduled DNA synthesis in permeable mouse sarcoma cells

2',3'-Dideoxythymidine triphosphate differentially inhibited replicative DNA synthesis in permeable mouse ascites sarcoma cells and unscheduled DNA synthesis in bleomycin-treated permeable cells or in isolated rat liver nuclei. The mode of inhibition of 2',3'-dideoxythymidine triphosphate was competitive with respect to deoxythymidine triphosphate. 2',3'-Dideoxythymidine triphosphate inhibited replicative DNA synthesis with a Ki of 8 microM, whereas unscheduled DNA synthesis was more sensitive, the Ki being 0.5 microM. Referring to the differential sensitivity of DNA polymerases alpha and beta to 2',3'-dideoxythymidine triphosphate and to other related information reported previously, the present results suggested that DNA polymerase alpha is playing a major role in replicative DNA synthesis, and DNA polymerase beta in unscheduled 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.     

Interactions of azidothymidine triphosphate with the cellular DNA polymerases alpha, delta, and epsilon and with DNA primase.

The interactions of azidothymidine triphosphate, the metabolically active form of the anti-AIDS drug azidothymidine (zidovudine), with the cellular DNA polymerases alpha, delta, and epsilon, as well as with the RNA primer-forming enzyme DNA primase were studied in vitro. DNA polymerase alpha was shown to incorporate azidothymidine monophosphate into a growing polynucleotide chain. This occurred 2000-fold slower than the incorporation of natural dTTP. Despite the ability of polymerase alpha to use azidothymidine triphosphate as an alternate substrate, this compound was only marginally inhibitory to the enzyme (Ki greater than 1 mM). Furthermore, the DNA primase activity associated with DNA polymerase alpha was barely inhibited by azidothymidine triphosphate (Ki greater than 1 mM). Inhibition was more pronounced for DNA polymerases delta and epsilon. The type of inhibition was competitive with respect to dTTP, with Ki values of 250 and 320 microM, respectively. No incorporation of azidothymidine monophosphate was detectable with these two DNA polymerases because their associated 3'- to 5'-exonuclease activities degraded primer molecules prior to any measurable elongation. Template-primer systems with a preformed 3'-azidothymidine-containing primer terminus inhibited the three replicative polymerases rather potently. DNA polymerase alpha was inhibited with a Ki of 150 nM and polymerases delta and epsilon with Ki values of 25 and 20 nM, respectively. The type of inhibition was competitive with respect to the unmodified substrate poly(dA).oligo(dT) for all DNA polymerases tested. Performed 3'-azidothymidine-containing primers hybridized to poly(dA) were rather resistant to degradation by the 3'- to 5'-exonuclease of DNA polymerases epsilon and more susceptible to the analogous activity that copurified with DNA polymerase delta. It is proposed that the repair of 3'-azidothymidine-containing primers might become rate-limiting for the process of DNA replication in cells that have been treated with azidothymidine triphosphate.     

Identification of DNA polymerase delta in CV-1 cells: studies implicating both DNA polymerase delta and DNA polymerase alpha in DNA replication

DNA polymerases delta and alpha were purified from CV-1 cells, and their sensitivities to the inhibitors aphidicolin, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), and monoclonal antibodies directed against DNA polymerase alpha were determined. The effects of these inhibitors on DNA replication in permeabilized CV-1 cells were studied to investigate the potential roles of polymerases delta and alpha in DNA replication. Aphidicolin was shown to be a more potent inhibitor of DNA replication than of DNA polymerase alpha or delta activity. Inhibition of DNA replication by various concentrations of BuPdGTP was intermediate between inhibition of purified polymerase alpha or delta activity. Concentrations of BuPdGTP which totally abolished DNA polymerase alpha activity were much less effective in reducing DNA replication, as well as the activity of DNA polymerase delta. Monoclonal antibodies which specifically inhibited polymerase alpha activity reduced, but did not abolish, DNA replication in permeable cells. BuPdGTP, as well as anti-polymerase alpha antibodies, inhibited DNA replication in a nonlinear manner as a function of time. Depending upon the initial or final rates of inhibition of replication by BuPdGTP and anti-alpha antibodies, as little as 50%, or as much as 80%, of the replication activity can be attributed to polymerase alpha. The remaining replication activity (20-50%) is tentatively attributed to polymerase delta, because it was aphidicolin sensitive and resistant to both anti-polymerase alpha antibodies and low concentrations of BuPdGTP. A concentration of BuPdGTP which abolished polymerase alpha activity reduced, but did not abolish, both the synthesis and maturation of nascent DNA fragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Evidence that DNA polymerases alpha and beta participate differentially in DNA repair synthesis induced by different agents

The roles of DNA polymerases alpha and beta in DNA replication and repair synthesis were studied in permeable animal cells, using different agents to induce repair synthesis. DNA polymerase inhibitors were used to investigate which polymerases were involved in repair synthesis and in replication. Polymerase alpha was responsible for replication. On the other hand, both polymerases alpha and beta were involved in DNA repair synthesis; the extent to which each polymerase participated depended primarily on the agent used to damage DNA. Polymerase beta was primarily responsible for repair synthesis induced by bleomycin or neocarzinostatin, whereas polymerase alpha played a more prominent role in repair synthesis indiced by N-methyl-N'-nitro-N-nitrosoguanidine or N-nitrosomethyl urea. More DNA damage was induced by the alkylating agents than by bleomycin or neocarzinostatin, suggesting that the extent of involvement of polymerase alpha or beta in DNA repair synthesis is related to the amount or type of DNA damage. In addition, salt concentration was found to have little or no effect on the results obtained with the DNA polymerase inhibitors. Our findings provide an explanation for conflicting reports in the literature concerning the roles of DNA polymerases alpha and beta in DNA repair.     

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.