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.     

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.     

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.     

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 polymerases required for repair of UV-induced damage in Saccharomyces cerevisiae.

The ability of yeast DNA polymerase mutant strains to carry out repair synthesis after UV irradiation was studied by analysis of postirradiation molecular weight changes in cellular DNA. Neither DNA polymerase alpha, delta, epsilon, nor Rev3 single mutants evidenced a defect in repair. A mutant defective in all four of these DNA polymerases, however, showed accumulation of single-strand breaks, indicating defective repair. Pairwise combination of polymerase mutations revealed a repair defect only in DNA polymerase delta and epsilon double mutants. The extent of repair in the double mutant was no greater than that in the quadruple mutant, suggesting that DNA polymerases alpha and Rev3p play very minor, if any, roles. Taken together, the data suggest that DNA polymerases delta and epsilon are both potentially able to perform repair synthesis and that in the absence of one, the other can efficiently substitute. Thus, two of the DNA polymerases involved in DNA replication are also involved in DNA repair, adding to the accumulating evidence that the two processes are coupled.     

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)     

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.     

Involvement of eucaryotic deoxyribonucleic acid polymerases alpha and gamma in the replication of cellular and viral deoxyribonucleic acid.

In an effort to identify the deoxyribonucleic acid (DNA) polymerase activities responsible for mammalian viral and cellular DNA replication, the effect of DNA synthesis inhibitors on isolated DNA polymerases was compared with their effects on viral and cellular DNA replication in vitro. DNA polymerase alpha, simian virus 40 (SV40) DNA replication in nuclear extracts, and CV-1 cell (the host for SV40) DNA replication in isolated nuclei all responded to DNA synthesis inhibitors in a quantitatively similar manner: they were relatively insensitive to 2',3'-dideoxythymidine 5'-triphosphate (d2TTP), but completely inhibited by aphidicolin, 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (araCTP), and N-ethylmaleimide. In comparison, DNA polymerases beta and gamma were inhibited by d2TTP but insensitive to aphidicolin and 20--30 times less sensitive to araCTP than DNA polymerase alpha. Herpes simplex virus type 1 (HSV-1) DNA polymerase and DNA polymerase alpha were the only enzymes tested that were relatively insensitive to d2TTP; DNA polymerases beta and gamma, phage T4 and T7 DNA polymerases, and Escherichia coli DNA polymerase I were 100--250 times more sensitive. The results with d2TTP were independent of enzyme concentration, primer-template concentration, primer-template choice, and the labeled dNTP. A specific requirement for DNA polymerase alpha in the replication of SV40 DNA was demonstrated by the fact that DNA polymerase alpha was required, in addition to other cytosol proteins, to reconstitute SV40 DNA replication activity in N-ethylmaleimide-inactivated nuclear extracts containing replicating SV40 chromosomes. DNA polymerases beta and gamma did not substitute for DNA polymerase alpha. In contrast to SV40 and CV-1 DNA replication, adenovirus type 2 (Ad-2) DNA replication in isolated nuclei was inhibited by d2TTP to the same extent as gamma-polymerase. Ad-2 DNA replication was also inhibited by aphidicolin to the same extent as alpha-polymerase. Synthesis of CV-1 DNA, SV40 DNA, and HSV-1 DNA in intact CV-1 cells was inhibited by aphidicolin. Ad-2 DNA replication was also inhibited, but only at a 100-fold higher concentration. We found no effect of 2'-3'-dideoxythymidine (d2Thd) on cellular or viral DNA replication in spite of the fact that Ad-2 DNA replication in isolated nuclei was inhibited 50% by a ratio of d2TTP/dTTP of 0.02. This was due to the inability of CV-1 and Hela cells to phosphorylate d2Thd to d2TTP. These data are consistent with the hypothesis that DNA polymerase alpha is the only DNA polymerase involved in replicating SV40 DNA and CV-1 DNA and that Ad-2 DNA replication involves both DNA polymerases gamma and alpha.     

Involvement of the essential yeast DNA polymerases in induced gene conversion

In the yeast Saccharomyces cerevisiae three different DNA polymerases alpha, delta and epsilon are involved in DNA replication. DNA polymerase alpha is responsible for initiation of DNA synthesis and polymerases delta and epsilon are required for elongation of DNA strand during replication. DNA polymerases delta and epsilon are also involved in DNA repair. In this work we studied the role of these three DNA polymerases in the process of recombinational synthesis. Using thermo-sensitive heteroallelic mutants in genes encoding DNA polymerases we studied their role in the process of induced gene conversion. Mutant strains were treated with mutagens, incubated under permissive or restrictive conditions and the numbers of convertants obtained were compared. A very high difference in the number of convertants between restrictive and permissive conditions was observed for polymerases alpha and delta, which suggests that these two polymerases play an important role in DNA synthesis during mitotic gene conversion. Marginal dependence of gene conversion on the activity of polymerase epsilon indicates that this DNA polymerase may be involved in this process but rather as an auxiliary enzyme.     

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)     

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.     

Replication of damaged DNA by translesion synthesis in human cells

Most types of DNA damage block the passage of the replication machinery. In order to bypass these blocks, cells employ special translesion synthesis (TLS) DNA polymerases, which have lower stringency than replicative polymerases. DNA polymerase eta is the major polymerase responsible for bypassing UV lesions in DNA and its absence results in the variant form of the genetic disorder, xeroderma pigmentosum. Other TLS polymerases have specificities for different types of damage, but their precise roles inside the cell have not yet been established. These polymerases are located in replication factories during DNA replication and the polymerase sliding clamp PCNA plays an important role in mediating switching between different polymerases.     

Human DNA polymerase beta initiates DNA synthesis during long-patch repair of reduced AP sites in DNA

Simple base damages are repaired through a short-patch base excision pathway where a single damaged nucleotide is removed and replaced. DNA polymerase beta (Pol beta) is responsible for the repair synthesis in this pathway and also removes a 5'-sugar phosphate residue by catalyzing a beta-elimination reaction. How ever, some DNA lesions that render deoxyribose resistant to beta-elimination are removed through a long-patch repair pathway that involves strand displacement synthesis and removal of the generated flap by specific endonuclease. Three human DNA polymerases (Pol beta, Pol delta and Pol epsilon) have been proposed to play a role in this pathway, however the identity of the polymerase involved and the polymerase selection mechanism are not clear. In repair reactions catalyzed by cell extracts we have used a substrate containing a reduced apurinic/apyrimidinic (AP) site resistant to beta-elimination and inhibitors that selectively affect different DNA polymerases. Using this approach we find that in human cell extracts Pol beta is the major DNA polymerase incorporating the first nucleotide during repair of reduced AP sites, thus initiating long-patch base excision repair synthesis.     

DNA polymerases as therapeutic targets

Numerous pathological states, including cancer, autoimmune diseases, and viral/bacterial infections, are often attributed to uncontrollable DNA replication. Inhibiting this essential biological process provides an obvious therapeutic target against these diseases. A logical target is the DNA polymerase, the enzyme responsible for catalyzing the addition of mononucleotides to a growing polymer using a DNA or RNA template as a guide for directing each incorporation event. This review provides a summary of therapeutic agents that target polymerase activity. A discussion of the biological function and mechanism of polymerases is first provided to illustrate the strategy for therapeutic intervention as well as the rational design of various nucleoside analogues that inhibit various polymerases associated with viral infections and cancer. The development of nucleoside and non-nucleoside inhibitors as antiviral agents is discussed with particular emphasis on their mechanism of action, structure-activity relationships, toxicity, and mechanism of resistance. In addition, commonly used anticancer agents are described to illustrate the similarities and differences associated with various nucleoside analogues as therapeutic agents. Finally, new therapeutic approaches that include the inhibition of selective polymerases involved in DNA repair and/or translesion DNA synthesis as anticancer agents are discussed.     

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 synthesis on discontinuous templates by human DNA polymerases: implications for non-homologous DNA recombination.

DNA polymerases catalyze the synthesis of DNA using a continuous uninterrupted template strand. However, it has been shown that a 3'-->5' exonuclease-deficient form of the Klenow fragment of Escherichia coli DNA polymerase I as well as DNA polymerase of Thermus aquaticus can synthesize DNA across two unlinked DNA templates. In this study, we used an oligonucleotide-based assay to show that discontinuous DNA synthesis was present in HeLa cell extracts. DNA synthesis inhibitor studies as well as fractionation of the extracts revealed that most of the discontinuous DNA synthesis was attributable to DNA polymerase alpha. Additionally, discontinuous DNA synthesis could be eliminated by incubation with an antibody that specifically neutralized DNA polymerase alpha activity. To test the relative efficiency of each nuclear DNA polymerase for discontinuous synthesis, equal amounts (as measured by DNA polymerase activity) of DNA polymerases alpha, beta, delta (+/- PCNA) and straightepsilon (+/- PCNA) were used in the discontinuous DNA synthesis assay. DNA polymerase alpha showed the most discontinuous DNA synthesis activity, although small but detectable levels were seen for DNA polymerases delta (+PCNA) and straightepsilon (- PCNA). Klenow fragment and DNA polymerase beta showed no discontinuous DNA synthesis, although at much higher amounts of each enzyme, discontinuous synthesis was seen for both. Discontinuous DNA synthesis by DNA polymerase alpha was seen with substrates containing 3 and 4 bp single-strand stretches of complementarity; however, little synthesis was seen with blunt substrates or with 1 bp stretches. The products formed from these experiments are structurally similar to that seen in vivo for non-homologous end joining in eukaryotic cells. These data suggest that DNA polymerase alpha may be able to rejoin double-strand breaks in vivo during replication.     

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.     

The overexpression of specialized DNA polymerases in cancer

Specialized DNA polymerases are required to bypass DNA damage lesions that would otherwise cause replication arrest and cell death. When operating on non-canonical templates, such as undamaged DNA or on non-cognate lesions, these polymerases exhibit considerably reduced fidelity, resulting in the generation of mutations. Ectopic overexpression of these polymerases can also lead to an increased mutation rate and an enhanced capability of DNA repair, suggesting that they could potentially act as oncogenes if they were overexpressed in cancers. Here, we examine expression patterns of DNA polymerases in matched normal and tumor samples from a diverse range of tissues. As well as investigating the specialized polymerases beta, lambda, iota and kappa, we also investigate the expression of the replicative polymerases alpha, delta and epsilon. The data presented provide evidence for the overexpression of specialized polymerases in tumors, with more than 45% of the 68 tumor samples studied demonstrating greater than two-fold enhanced expression of at least one specialized polymerase. Of particular note, DNA polymerase beta (pol beta) was found to be overexpressed at both the mRNA and protein level in approximately one third of all tumor types studied, with overexpression being particularly frequent in uterus, ovary, prostate and stomach samples. Pols lambda, and iota were also found to be overexpressed to a significant extent in a range of tumor types, albeit less frequently than pol beta. In contrast, pol kappa was rarely found to be overexpressed in tumors but was found to be commonly underexpressed in many samples. Downregulation of pol beta expression by siRNA resulted in an increased sensitivity to the chemotherapeutic agent cisplatin, suggesting a role for this polymerase in providing tolerance to cisplatin-induced damage. These observations suggest that specialised DNA polymerases, and particularly pol beta, could be considered both as caretaker genes altered during tumorigenesis, and as potential drug targets to sensitise tumors to chemotherapy.     

Aphidicolin inhibition of the production of replicative-form DNA during bovine parvovirus infection.

Since parvoviruses apparently do not possess a DNA polymerase activity, one or more of the host cell DNA polymerases must be responsible for replicating the single-stranded DNA genome. We have focused on determining which polymerase, alpha, beta, or gamma (pol alpha, pol beta, or pol gamma, respectively), is responsible for the first step in bovine parvoviral DNA replication: conversion of the single-stranded DNA genome to a parental replicative form (RF). In this study, we used aphidicolin, a specific inhibitor of DNA pol alpha, to assay for the requirement of pol alpha activity in parental RF formation in vivo. Synchronized cell cultures were infected with bovine parvovirus with or without aphidicolin, and the products of viral replication were separated on agarose gels and identified by Southern blot analysis. We found that complete inhibition of viral DNA synthesis resulted when 20 microM aphidicolin was present throughout the infection. In addition, viral DNA synthesis was inhibited by as little as 1 microM aphidicolin, whereas lower concentrations (0.1 and 0.01 microM) resulted in partial inhibition of the replication process. Using 32P-labeled bovine parvovirus as the input virus we differentiated parental RF from daughter RF and progeny DNA synthesis. We conclude that DNA pol alpha is required for the production of RF during bovine parvovirus replication in vivo and that this requirement is most likely for the conversion of bovine parvovirus input single-stranded DNA to parental RF. These results do not rule out a possible role for DNA pol gamma in the first step, nor do they rule out a role for pol alpha or pol gamma in later stages of the replication cycle.