The yeast environmental stress response regulates mutagenesis induced by proteotoxic stress.

Conditions of chronic stress are associated with genetic instability in many organisms, but the roles of stress responses in mutagenesis have so far been elucidated only in bacteria. Here, we present data demonstrating that the environmental stress response (ESR) in yeast functions in mutagenesis induced by proteotoxic stress. We show that the drug canavanine causes proteotoxic stress, activates the ESR, and induces mutagenesis at several loci in an ESR-dependent manner. Canavanine-induced mutagenesis also involves translesion DNA polymerases Rev1 and Polζ and non-homologous end joining factor Ku. Furthermore, under conditions of chronic sub-lethal canavanine stress, deletions of Rev1, Polζ, and Ku-encoding genes exhibit genetic interactions with ESR mutants indicative of ESR regulating these mutagenic DNA repair processes. Analyses of mutagenesis induced by several different stresses showed that the ESR specifically modulates mutagenesis induced by proteotoxic stress. Together, these results document the first known example of an involvement of a eukaryotic stress response pathway in mutagenesis and have important implications for mechanisms of evolution, carcinogenesis, and emergence of drug-resistant pathogens and chemotherapy-resistant tumors.

Inhibitory effects of sword bean extract on alveolar bone resorption induced in rats by Porphyromonas gingivalis infection.

BACKGROUND: The domesticated legume, Canavalia gladiata (commonly called the sword bean), is known to contain canavanine. The fruit is used in Chinese and Japanese herbal medicine for treating the discharge of pus, but its pharmacological mechanisms are still unclear. OBJECTIVES: This study examined the effect of sword bean extract (SBE) on (i) oral bacteria and human oral epithelial cells in vitro, and (ii) the initiation and progression of experimental Porphyromonas gingivalis-induced alveolar bone resorption in rats. MATERIAL AND METHODS: A high-performance liquid chromatography/ultraviolet method was applied to quantitate canavanine in SBE. By assessing oral bacterial growth, we estimated the minimum inhibitory concentration and minimum bactericidal concentration of SBE, canavanine, chlorhexidine gluconate (CHX) solution. The cytotoxicity of SBE, canavanine, CHX, leupeptin and cystatin for KB cells was determined using a trypan blue assay. The effects of SBE, canavanine, leupeptin and cystatin on Arg-gingipain (Rgp) and Lys-gingipain (Kgp) were evaluated by colorimetric assay using synthetic substrates. To examine its effects on P. gingivalis-associated periodontal tissue breakdown, SBE was orally administered to P. gingivalis-infected rats. RESULT: Sword bean extract contained 6.4% canavanine. SBE and canavanine inhibited the growth of P. gingivalis and Fusobacterium nucleatum. The cytotoxicity of SBE, canavanine and cystatin on KB cells was significantly lower than that of CHX. Inhibition of Rgp with SBE was comparable to that with leupeptin, a known Rgp inhibitor, and inhibition of Kgp with SBE was significantly higher than that with leupeptin at 500 µg/mL ( p < 0.05). P. gingivalis-induced alveolar bone resorption was significantly suppressed by administration of SBE, with bone levels remaining comparable to non-infected animals ( p < 0.05). CONCLUSION: The present study suggests that SBE might be effective against P. gingivalis-associated alveolar bone resorption.

Quantification of canavanine, 2-aminoethanol, and cyanamide in Aphis craccivora and its host plants, Robinia pseudoacacia and Vicia angustifolia: effects of these compounds on larval survivorship of Harmonia axyridis.

The cowpea aphid Aphis craccivora that infests the black locust Robinia pseudoacacia shows toxicity to its predator, the multicolored Asian ladybird beetle, Harmonia axyridis. In contrast, the same aphid species that infests the common vetch, Vicia angustifolia, is suitable prey for H. axyridis larvae. Previously, it was reported that the toxicity of A. craccivora infesting R. pseudoacacia was due to canavanine and 2-aminoethanol, but there was some doubt about the toxicity of these compounds and their concentrations in the aphids. In the present study, we determined the concentrations of cyanamide, canavanine, and 2-aminoethanol in A. craccivora infesting the two host plants. In the extracts of A. craccivora that infested either of the host plants, canavanine was undetectable, and 2-aminoethanol was detected at the concentration of 3.0-4.0 µg/g fresh weight. Cyanamide was detected in the extract of A. craccivora that infested R. pseudoacacia (7.7 µg/g fresh weight) but not in that infesting V. angustifolia. The toxicity of canavanine, 2-aminoethanol, and cyanamide was evaluated against H. axyridis larvae in a bioassay by using an artificial diet containing these compounds at various concentrations. Cyanamide exhibited 10-100 times stronger toxicity than canavanine and 2-aminoethanol. These results indicate that the toxicity is at least partly due to cyanamide, which is present in the toxic A. craccivora that infests R. pseudoacacia but absent from the non-toxic A. craccivora that infests V. angustifolia.

Amino acid analog toxicity in primary rat neuronal and astrocyte cultures: implications for protein misfolding and TDP-43 regulation.

Amino acid analogs promote translational errors that result in aberrant protein synthesis and have been used to understand the effects of protein misfolding in a variety of physiological and pathological settings. TDP-43 is a protein that is linked to protein aggregation and toxicity in a variety of neurodegenerative diseases. This study exposed primary rat neurons and astrocyte cultures to established amino acid analogs (canavanine and azetidine-2-carboxylic acid) and showed that both cell types undergo a dose-dependent increase in toxicity, with neurons exhibiting a greater degree of toxicity compared with astrocytes. Neurons and astrocytes exhibited similar increases in ubiquitinated and oxidized protein following analog treatment. Analog treatment increased heat shock protein (Hsp) levels in both neurons and astrocytes. In neurons, and to a lesser extent astrocytes, the levels of TDP-43 increased in response to analog treatment. Taken together, these data indicate that neurons exhibit preferential toxicity and alterations in TDP-43 in response to increased protein misfolding compared with astrocytes.

Misincorporation of amino acid analogues into proteins by biosynthesis.

Despite astounding diversity in their structure and function, proteins are constructed from 22 protein or 'canonical' amino acids. Hundreds of amino acid analogues exist; many occur naturally in plants, some are synthetically produced or can be produced in vivo by oxidation of amino acid side-chains. Certain structural analogues of the protein amino acids can escape detection by the cellular machinery for protein synthesis and become misincorporated into the growing polypeptide chain of proteins to generate non-native proteins. In this review we seek to provide a comprehensive overview of the current knowledge on the biosynthetic incorporation of amino acid analogues into proteins by mammalian cells. We highlight factors influencing their incorporation and how the non-native proteins generated can alter cell function. We examine the ability of amino acid analogues, representing those commonly found in damaged proteins in pathological tissues, to be misincorporated into proteins by cells in vitro, providing us with a useful tool in the laboratory to generate modified proteins representing those present in a wide-range of pathologies. We also discuss the evidence for amino acid analogue incorporation in vivo and its association with autoimmune symptoms. We confine the review to studies in which the synthetic machinery of cell has not been modified to accept non-protein amino acids.

High-throughput transformation of Saccharomyces cerevisiae using liquid handling robots.

Saccharomyces cerevisiae (budding yeast) is a powerful eukaryotic model organism ideally suited to high-throughput genetic analyses, which time and again has yielded insights that further our understanding of cell biology processes conserved in humans. Lithium Acetate (LiAc) transformation of yeast with DNA for the purposes of exogenous protein expression (e.g., plasmids) or genome mutation (e.g., gene mutation, deletion, epitope tagging) is a useful and long established method. However, a reliable and optimized high throughput transformation protocol that runs almost no risk of human error has not been described in the literature. Here, we describe such a method that is broadly transferable to most liquid handling high-throughput robotic platforms, which are now commonplace in academic and industry settings. Using our optimized method, we are able to comfortably transform approximately 1200 individual strains per day, allowing complete transformation of typical genomic yeast libraries within 6 days. In addition, use of our protocol for gene knockout purposes also provides a potentially quicker, easier and more cost-effective approach to generating collections of double mutants than the popular and elegant synthetic genetic array methodology. In summary, our methodology will be of significant use to anyone interested in high throughput molecular and/or genetic analysis of yeast.

Growth inhibition of a rat colon tumor by L-canavanine.

The effects of L-canavanine, a higher plant nonprotein amino acid, on the growth of a rat colon carcinoma were assessed. The 1 and 10% lethal dose values following a single s.c. injection in Fischer rats were 4.75 and 5.57 g/kg, respectively. Rats received s.c. injections of a 10% (w/v) tumor cell suspension. When the tumors reached a size of 500 to 1000 mm3, the rats received canavanine, 2.0 g/kg or 3.0 g/kg s.c. daily for 5 or daily for 9 days. Control animals received a 0.9% NaCl solution. Administration of canavanine, 2.0 g/kg for 5 days produced a treated versus control of 23%; the treated versus control for 9 days was 14%. The 3.0-g/kg dosing regimen resulted in a treated versus control value of -13% after 5 days and -8% after 9 days. The negative values indicated regression of the tumor. The reduction in tumor volume, expressed as the percentage of regression, was 22% in animals receiving canavanine, 3.0 g/kg daily for 5 days and 60% in the 3.0-g/kg-daily-for-9-days treatment group. Cumulative toxicity caused death in 2 of 5 animals in the 3.0-g/kg-for-9-days treatment group; the average weight loss was 31%. The 3.0-g/kg-for-5-days treatment also produced undesirable cumulative toxicity as indicated by a weight loss of 19%. Cumulative toxicity was reduced greatly when canavanine was administered at a dose level of 2.0 g/kg for 5 days (weight loss of 13%). Analysis of the relationship of caloric deprivation to tumor growth reduction established that canavanine-mediated curtailment of tumor growth was not caused by reduced food intake and its associated loss in body weight. Histological examination of tissues from rats receiving canavanine, 2.0 or 3.0 g/kg daily for 5 or 9 days failed to reveal lesions in any of the examined tissues, except for varying degrees of pancreatic acinar atrophy. All other tissues appeared normal. The white and red blood cell values of canavanine-treated rats were also normal following 1, 3, or 6 injections of canavanine, 2.0 or 3.0 g/kg. The results indicated that canavanine induced marked growth inhibition of the rat colon carcinoma. Our experiments also disclosed that further studies must be conducted to optimize the dosing schedule to enhance drug efficacy and to reduce its cumulative toxicity.

Enhancement of human tumor cell killing by L-canavanine in combination with gamma-radiation.

On the basis of several physiological properties of L-canavanine, we have tested the prediction that this analogue of arginine would enhance the cytotoxic effects of gamma-rays in mammalian cells. Using the human colonic tumor cell line, HT-29, time-dose studies were performed with log-phase cultures in order to determine conditions which maximize the incorporation of L-canavanine into cellular proteins while leaving a large fraction of the cells viable for subsequent gamma-ray survival measurements. At an input ratio of 2.5 (L-canavanine:arginine), the analogue exerted a cytostatic effect on the cells for at least 6 days following one cell division. Little cell killing (less than 20%) by clonogenicity was caused by L-canavanine during the first 12 hr of treatment of log-phase cells, even at a L-canavanine:arginine ratio of 20. A 24-hr exposure, however, produced an exponential decrease in survival as a function of L-canavanine concentration. The interaction between L-canavanine treatment and gamma-ray damage with respect to cell survival was examined under several conditions and times based on the above findings. Optimal enhancement of X-ray-induced cytotoxicity (assayed by loss of clonogenicity) was observed with a 48-hr exposure to the analogue at a L-canavanine:arginine ratio of 10. A marked increase in radiosensitivity was observed when L-canavanine was administered either before or after irradiation of the cells. In both protocols, enhancement was seen at all radiation doses. Together with our earlier findings showing the antitumor activity of L-canavanine in L1210 murine leukemia, these results suggest the potential usefulness of this amino acid analogue in the treatment of cancer.

African herbal medicines in the treatment of HIV: Hypoxis and Sutherlandia. An overview of evidence and pharmacology.

In Africa, herbal medicines are often used as primary treatment for HIV/AIDS and for HIV-related problems. In general, traditional medicines are not well researched, and are poorly regulated. We review the evidence and safety concerns related to the use of two specific African herbals, which are currently recommended by the Ministry of Health in South Africa and member states for use in HIV: African Potato and Sutherlandia. We review the pharmacology, toxicology and pharmacokinetics of these herbal medicines. Despite the popularity of their use and the support of Ministries of Health and NGOs in some African countries, no clinical trials of efficacy exist, and low-level evidence of harm identifies the potential for drug interactions with antiretroviral drugs. Efforts should be made by mainstream health professionals to provide validated information to traditional healers and patients on the judicious use of herbal remedies. This may reduce harm through failed expectations, pharmacologic adverse events including possible drug/herb interactions and unnecessary added therapeutic costs. Efforts should also be directed at evaluating the possible benefits of natural products in HIV/AIDS treatment.

Bacteria do not incorporate ß-N-methylamino-L-alanine into their proteins.

ß-N-methylamino-l-alanine (BMAA), is commonly found in both a free and proteinassociated form in various organisms exposed to the toxin. The long latency of development of neurodegeneration attributed to BMAA, is hypothesized to be the result of excitotoxicity following slow release of the toxin from protein reservoirs. It was recently suggested that these BMAA-protein associations may reflect misincorporation of BMAA in place of serine, as occurs, for example, when canavanine misincorporates in place of arginine. We therefore compared BMAA and canavanine toxicty in various bacterial species, and misincorporation of these amino acids into proteins in a bacterial protein expression system. None of the bacterial species showed any physiological stress responses to BMAA in contrast to the growth reduction observed when cultures were incubated in media containing canavanine. LC-MS analysis confirmed uptake of BMAA from growth media. However, after immobilized metal affinity chromatography and SDS-PAGE purification of proteins produced in an E scherichia coli expression system, no BMAA was detected by either LC-MS or LC-MS/MS analysis using two derivatization methods, or by orbitrap MS of trypsin digests of the protein. We therefore conclude that BMAA is not misincorporated into proteins in bacteria and that the observed BMAA-protein association in bacteria is superficial.

Selective cytotoxicity of L-canavanine in tumorigenic Madin-Darby canine kidney T1 cells.

L-canavanine, an analog of L-arginine, was examined for toxicity in a normal canine kidney epithelial cell line, Madin-Darby canine kidney (MDCK), and in its chemically transformed derivative MDCK-T1. Under conditions where the analog reversibly arrested growth of MDCK cells, more than 90% of the tumorigenic cells were killed. This selective cytotoxicity was not due to any difference in growth rate (both lines doubled every 24 h). Nor was it caused by the inhibition of protein synthesis or DNA replication, since amino acid deprivation and drugs which inhibited replication directly and indirectly did not kill the transformed cells preferentially. To the contrary, cycloheximide killed MDCK cells preferentially. Although the mechanism for the selective cytotoxicity of canavanine in the tumorigenic cells remains obscure, one clue was afforded by the observation that the analog caused a loss in plating efficiency of the T1 cells prior to their detachment from plates. Selective sensitivity to canavanine may therefore reside in cell surface proteins, which are known to differ both qualitatively and quantitatively between normal and transformed cells. The present results support our previous findings suggesting the possible value of using canavanine as an agent for cancer chemotherapy.

Elimination of toxicity from diets containing alfalfa seeds.

Cynomolgus macaques were fed autoclaved alfalfa seeds for up to 1 yr. There were no humoral signs of a syndrome resembling systemic lupus erythematosus. The data are in contrast to those previously reported in monkeys fed raw alfalfa seeds, in which a systemic lupus erythematosus-like syndrome was induced in a shorter interval. The autoclaved seeds retained antihypercholesterolaemic effects.

The effect of food chemicals on cell aging of human diploid cells in in vitro culture.

The potency of food chemicals to induce cell aging was evaluated in human diploid fibroblast cells HAIN-55 having a finite replicative potential by using in vitro aging markers, i.e., decreases of maximum proliferative potential (lifespan) of cells, saturation density in monolayer culture (SD), plating efficiency (PE) and mitotic index (MI), and an increase of cells with polyploid karyotypes. By treatment twice with low concentration of genotoxic chemicals aflatoxin B1, allylisothiocyanate or trans-cinnamaldehyde (severe clastogenic flavoring agent; Kasamaki et al., 1982), lifespan (expressed by the number of cumulative cell population doubling (CPD)) of the treated cells was reduced by 8-12 CPDs accompanied by change of the other aging markers. By successive treatment (29 or 25 times) with non-genotoxic chemical aspartame (N-L-aspartyl-L-phenylalanine) or L-canavanine (structural analogue of L-arginine), lifespan of the treated cells was also slightly shortened (by 2-6 CPDs) compared with the untreated control cells. In the process of cell aging, Mitochondrial activity (MTT activity) decreased almost in parallel with the decrease of SD and MI. On the basis of these results, a variety of genotoxic and non-genotoxic chemicals were examined by using MTT activity as the aging marker for their effects on the aging of HAIN-55 cells and bovine artery endothelial cells which also had a finite replicative potential. The results showed that seven genotoxic and nine non-genotoxic chemicals promoted cell aging.

[Isolation and characteristics of new mutants of Saccharomyces cerevisiae with increased spontaneous mutability]. / Vydelenie i kharakteristika novykh mutantov drozhzhei Saccharomyces cerevisiae s povyshennoi spontannoi mutabil'nost'iu.

To isolate some new genes controlling the process of spontaneous mutagenesis, a collection of 16 yeast strains with enhanced rate of spontaneous canavanine resistant mutations was obtained. Genetical analysis allowed to define that the mutator phenotype of these strains is due to a single nuclear mutation. Such mutations were called hsm (high spontaneous mutagenesis). Recombinational test showed that 5 mutants under study carried 5 nonallelic mutations. It was revealed that the mutation hsm3-1 is a nonspecific mutator elevating the rate of both spontaneous canavanine resistant mutations and the frequency of reversions in mutations lys1-1 and his1-7. Genetical analysis revealed that mutation hsm3-1 is recessive. The study of cross sensitivity of mutator strains to physical and chemical mutagens demonstrated that 12 of 16 hsm mutants were resistant to the lethal action of UV, gamma rays and methylmethanesulfonate, and 4 mutants were only sensitive to these factors. Possible nature of hsm mutations is discussed.

Amino acid discrimination by the nuclear encoded mitochondrial arginyl-tRNA synthetase of the larva of a bruchid beetle (Caryedes brasiliensis) from northwestern Costa Rica.

L-canavanine, the toxic guanidinooxy analogue of L-arginine, is the product of plant secondary metabolism. The need for a detoxifying mechanism for the producer plant is self-evident but the larvae of the bruchid beetle Caryedes brasiliensis, that is itself a non-producer, have specialized in feeding on the Lcanavanine-containing seeds of Dioclea megacarpa. The evolution of a seed predator that can imitate the enzymatic abilities of the host permits us to address the question of whether the same problem of amino acid recognition in two different kingdoms has been solved by the same mechanism. A discriminating arginyl-tRNA synthetase, detected in a crude C. brasiliensis larval extract, was proposed to be responsible for insect's ability to survive the diet of L-canavanine (Rosenthal, G. A., Dahlman, D. L., and Janzen, D. H. (1976) A novel means for dealing with L-canavanine, a toxic metabolite. Science 192, 256e258). Since the arginyl-tRNA synthetase of at least three genetic compartments (insect cytoplasmic, insect mitochondrial and insect gut microflora) may participate in conferring L-canavanine resistance, we investigated whether the nuclear-encoded C. brasiliensis mitochondrial arginyl-tRNA synthetase plays a role in this discrimination. Steady state kinetics of the cloned, recombinant enzyme have revealed and quantified an amino acid discriminating potential of the mitochondrial enzyme that is sufficient to account for the overall L-canavanine misincorporation rate observed in vivo. As in the cytoplasmic enzyme of the L-canavanine producer plant, the mitochondrial arginyl-tRNA synthetases from a specialist seed predator relies on a kinetic discrimination that prevents L-canavanine misincorporation into proteins.

Metabolism of L-[guanidinooxy-14C]canavanine in the rat.

The metabolism of L-canavanine, a nonprotein amino acid with significant antitumor effects, was investigated. L-Canavanine, provided at 2.0 g/kg, was supplemented with 5 microCi of L-[guanidinooxy-14C]canavanine (58 microCi/mumol) and administered iv, sc, or orally to female Sprague-Dawley rats weighing approximately 200 g. 14C recovery in the urine at 24 hr was 83, 68, or 61%, respectively, of the administered dose. Another 5-8% of the 14C was expired as 14CO2. The gastrointestinal tract contained 21% of orally administered 14C. Serum, feces, tissues, and de novo synthesized proteins only accounted for a few percent of the original dose by any administrative route. Analysis of the 14C-containing urinary metabolites revealed that [14C] urea accounted for 88% of the urinary radioactivity for an iv injection, 75% for sc administration, and 50% following an oral dose. By all routes of administration, [14C]guanidine represented 5% of the radioactivity in the urine and [14C]guanidinoacetic acid accounted for 2%. Serum and urine amino acid analysis showed a markedly elevated ornithine level. Basic amino acids such as histidine, lysine, and arginine were also higher in the urine. Plasma ammonia levels were determined following oral canavanine doses of 1.0, 2.0, and 4.0 g/kg. A rapid but transient elevation in plasma ammonia was observed only at the 4.0 g/kg dose. This indicates that elevated plasma ammonia is not a likely cause of canavanine toxicity at the drug concentrations used in this study.

Growth inhibition of A549 human lung adenocarcinoma cells by L-canavanine is associated with p21/WAF1 induction.

L-Canavanine (CAV) is a higher plant nonprotein amino acid and a potent L-arginine antimetabolite. CAV can inhibit the proliferation of tumor cells in vitro and in vivo, but little is known regarding the molecular mechanisms mediating these effects. We demonstrated that the treatment of human lung adenocarcinoma A549 cells with CAV caused growth inhibition; G1 phase arrest is accompanied by accumulation of an incompletely phosphorylated form of the retinoblastoma protein, whose phosphorylation is necessary for cell cycle progression from G1 to S phase. In addition, CAV induces the expression of p53 and subsequent expression of a cyclin-dependent kinase inhibitor, p21/WAF1. The p53-dependent induction of p21/WAF1 and the following dephosphorylation of the retinoblastoma protein by CAV could account for the observed CAV-mediated G1 phase arrest.

The biochemical basis for L-canavanine tolerance by the tobacco budworm Heliothis virescens (Noctuidae).

The tobacco budworm, Heliothis virescens (Noctuidae), a destructive insect pest, is remarkably resistant to L-canavanine, L-2-amino-4-(guanidinooxy)butyric acid, an arginine antimetabolite that is a potent insecticide for nonadapted species. H. virescens employs a constitutive enzyme of the larval gut, known trivially as canavanine hydrolase (CH), to catalyze an irreversible hydrolysis of L-canavanine to L-homoserine and hydroxyguanidine. As such, it represents a new type of hydrolase, one acting on oxygen-nitrogen bonds (EC 3.13.1.1). This enzyme has been isolated from the excised gut of H. virescens and purified to homogeneity; it exhibits an apparent Km value for L-canavanine of 1.1 mM and a turnover number of 21.1 micromol x min(-1)x micromol(-1). This enzyme has a mass of 285 kDa and is composed of two subunits with a mass of 50 kDa or 47.5 kDa. CH has a high degree of specificity for L-canavanine as it cannot function effectively with either L-2-amino-5-(guanidinooxy)pentanoate or L-2-amino-3-(guanidinooxy)propionate, the higher or lower homolog of L-canavanine, respectively. L-Canavanine derivatives such as methyl-L-canavanine, or L-canaline and O-ureido-L-homoserine, are not metabolized significantly by CH.

Reversible inhibition of bovine parvovirus DNA replication by aphidicolin and L-canavanine.

The replication of the autonomous parvovirus, bovine parvovirus (BPV), has been studied in virus-infected cells. Gel electrophoresis was used to determine the effect of aphidicolin, a specific inhibitor of DNA polymerase alpha, and L-canavanine, an inhibitor of protein synthesis, on viral DNA replication. Synchronized cell cultures were infected with 32P-labelled or unlabelled BPV in the presence or absence of aphidicolin and L-canavanine. Cells were harvested at various times post-infection, and DNA was electrophoresed and blotted. When aphidicolin was added to cells at the time of infection, then removed 8 h later, BPV replicative form DNA (RF) synthesis began within 2 h after its removal. This preceded the peak of cellular DNA synthesis by 2 h, unlike an uninhibited infection, when viral RF synthesis follows the peak of S phase by 2 to 4 h. Furthermore, if aphidicolin was added at any point during the replication cycle, BPV DNA synthesis stopped. This effect was shown to be completely reversible and indicated that aphidicolin did not disrupt the replication apparatus required for viral DNA synthesis. L-Canavanine inhibited synthesis of the virus-specific proteins NP-1 and VP3 and synthesis of BPV DNA. Upon removal of L-canavanine, viral protein synthesis was detected by 30 min followed by viral DNA synthesis. These results indicate that a specific S phase function other than cellular DNA synthesis is required for initiation of BPV DNA synthesis, that DNA polymerase alpha plays a major role in BPV DNA replication in vivo, and that these inhibitors can be used to inhibit reversibly various stages of BPV DNA replication.