Repair of lesions induced by bruneomycin in DNA of isolated mitochondria from the mature eggs of the teleost fish Misgurnus fossilis

Addition of a radiomimetic antibiotic bruneomycin (Streptonigrin) to isolated mitochondria from mature quiescent oocytes of the teleost fish loach Misgurnus fossilis leads to the induction of unscheduled synthesis of mitochondrial DNA. Most of the newly synthesized DNA has the sedimentation properties of open circles and up to 15% of the label is present in the fraction of the covalently closed-circular molecules. The size of the newly synthesized DNA stretches determined from the bouyant shift of DNA labeled with 5-bromouracil and [3H]dAMP and sonicated to fragments of different molecular weight, was found to be equal to about 1000 nucleotides for the labeled covalently closed circles and to about 2000 nucleotides for the labeled open-circular DNA. Experiments with the centrifugation of non-sheared and sonicated 5-bromouracil and [3H]dAMP-labeled mitochondrial DNA (mtDNA) in alkaline CsCl density gradients provided evidence of a covalent linkage between newly-synthesized stretches and the parental DNA strands. It is concluded from these data that the unscheduled mtDNA synthesis induced by bruneomycin does at least in part represent mtDNA repair synthesis.     

Potentiation of sulphur mustard or cisplatin-induced toxicity by caffeine in Chinese hamster cells correlates with formation of DNA double-strand breaks during replication on a damaged template

Caffeine inhibited the elongation of nascent DNA and induced breaks in the template DNA of sulphur mustard-treated Chinese hamster cells. The sizes of template and nascent DNAs, as indicated by alkaline sucrose gradient sedimentation, were similar suggesting that incision of template DNA occurred opposite gaps formed in nascent DNA by the action of caffeine, forming, effectively, double-strand breaks in DNA. Double-strand break formation was demonstrated, by means of elution of labelled DNA through polycarbonate filters at neutral pH, in both sulphur mustard- and cisplatin-treated cells when they were incubated in the presence of caffeine for 24 h. Double-strand breaks were only formed in that DNA which had been replicated in the presence of caffeine after treatment with sulphur mustard or cisplatin. Non-toxic concentrations of cycloheximide abolished the potentiation by caffeine of sulphur mustard-induced toxicity to Chinese hamster cells and at the same time abolished the formation of the low molecular weight nascent DNA, and as a consequence of its inhibitory effect on DNA synthesis, and the formation of double-strand breaks in DNA. Potentiation of the lethal and clastogenic effects of genotoxic agents by caffeine is therefore due to effects on the rate and mode of DNA synthesis which lead finally to double-strand breaks in DNA.     

Purification and characterization of covalently closed replicative intermediates of ColEl DNA from Escherichia coli.

Pulse-labeled ColEl DNA molecules, undergoing replication in Escherichia coli cells either in the absence or presence of chloramphenicol, were extracted and purified by neutral sucrose density gradient sedimentation and equilibrium centrifugation in an ethidium bromide-cesium chloride gradient. In the dye-buoyant density gradient, the replicating molecules were found in regions between the supercoiled and open-circular nonreplicating plasmid DNA, as well as in the open-circular region. In a neutral sucrose gradient, peaks of pulse label were found in the region of 26 to 38 S as well as at the 23 and 17 S positions corresponding to the positions of supercoiled and open-circular ColEl DNA. In alkaline sucrose gradient, nascent ColEl DNA was found to sediment as discrete peaks corresponding to 5-6, 7-9, and 14-16 S, indicating that at least one growing strand of the replicating molecule is produced discontinuously. In the electron microscope, many of the molecules appeared as partially supercoiled structures containing two open-circular branches of equal length, of less than 20% to more than 90% replicated. Branched open-circular molecules were not observed to any significant extent without prior treatment to induce single-strand scissions. The parental strands of the replicating molecules were determined to be covalently closed, but the superhelical density of the DNA was shown to be progressively decreased as replication proceeded.     

Isolation and characterization of a DNA primase from human mitochondria

A family of enzymatic activities isolated from human mitochondria is capable of initiating DNA replication on single-stranded templates. The principal enzymes include at least a primase and DNA polymerase gamma and require that rNTPs as well as dNTPs be present in the reaction mixture. Poly(dC) and poly(dT), as well as M13 phage DNA, are excellent templates for the primase activity. A single-stranded DNA containing the cloned origin of mitochondrial light-strand synthesis can be a more efficient template than M13 phage DNA alone. Primase and DNA polymerase activities were separated from each other by sedimentation in a glycerol density gradient. Using M13 phage DNA as template, these mitochondrial enzymes synthesize RNA primers that are 9 to 12 nucleotides in size and are covalently linked to nascent DNA. The formation of primers appears to be the rate-limiting step in the replication process. Replication of M13 DNA is sensitive to N-ethylmaleimide and dideoxynucleoside triphosphates, but insensitive to rifampicin, alpha-amanitin, and aphidicolin.     

Presence of two deoxyribonucleic acid polymerases in bull spermatozoa.

A DNA polymerase-endogenous template complex was isolated from nuclear heads of bull spermatozoa. The buoyant density of the complex was 1.15 g/cm 3. The sedimentation coefficient of the nuclear DNA polymerase isolated from the complex was higher at low ionic strength, but approached 3.4S when centrifuged in a medium containing 2M-KCl. Activated exogenous DNA increased polymerase activity. Only very low activities were detected with synthetic templates such as poly(A).(dT)12-18 and poly(dT).poly(A). The nuclear reaction was stimulated by 150mM-KCl and was slightly inhibited by N-ethylmaleimide; it was resistant to actinomycin D, netropsin and ethidium bromide. Another DNA polymerase, highly sensitive to ethidium bromide, was extracted from the mitochondira-rich middle-piece fraction. Its sedimentation coefficient was close to 9S, but fell to approx. 4S in high-ionic-strength medium.     

Nature of the Complementary Strands Synthesized in Vitro upon the Single-Stranded Circular DNA of Bacteriophage øX174 after Ultraviolet Irradiation

This paper describes experiments intended to decide whether UV lesions in DNA act as absolute blocks to chain elongation by the Escherichia coli DNA polymerase or only slow down the polymerization process. Ultraviolet (UV)-irradiated, single-stranded (SS) circular DNA of bacteriophage øX174 was used as template for the polymerase in a reaction mixture in vitro, under conditions allowing synthesis of not more than one complementary strand per template molecule. The mean length of the newly synthesized complementary strands (as determined by velocity sedimentation in alkaline CsCl gradients), as well as the over-all template activity (as measured by deoxyadenosine monophosphate [dAMP] incorporation) was found to decrease with the number of biologically lethal hits sustained by the irradiated templates. With the increase of time or temperature of reaction, the net synthesis of complementary strands increased (as a consequence of increased initiation), but their mean length remained constant. The mean length of synthesized strands was greater than would be expected if all biologically lethal hits were to block the polymerization process. The lethal hits which serve as blocking lesions are inferred to be pyrimidine dimers because it is possible to obtain synthesis of full-length complementary strands if, when heat-denatured, UV-irradiated, double-stranded replicative form (RF II) DNA of bacteriophage øX174 is used as a template, it is pretreated with yeast photoreactivating enzyme (YPRE) in presence of visible light.     

ACCUMULATION OF REPLICATIVE INTERMEDIATES OF MITOCHONDRIAL DNA IN TETRAHYMENA PYRIFORMIS GROWN IN ETHIDIUM BROMIDE

The effect of growth of Tetrahymena pyriformis in ethidium bromide (EthBr) on the structure and synthesis of mitochondrial DNA (mtDNA) has been investigated. During the first 5 h of growth in EthBr, mtDNA synthesis is inhibited 95% or more. After 10–15 h, this block is partially released and large numbers of replicating molecules accumulate, indicating that inhibition by EthBr primarily affects the rate of chain growth and not the initiation of new rounds of replication. The accumulated molecules sediment more rapidly than normal Tetrahymena mtDNA and do not contain enough single-strand regions to distinguish them from normal Tetrahymena mtDNA when banded in buoyant CsCl or NaI gradients. Electron microscopy shows that the predominant species in this rapidly sedimenting DNA is a linear molecule containing one symmetrical double-stranded replication loop of varying size located at its center. No degradation of mtDNA from cells grown in EthBr was detected in alkaline velocity gradients.     

Differentiation and characterization of the cytoplasmic and nuclear deoxyribonucleic acid polymerases from baby hamster kidney cells.

Distinct DNA polymerase activities have been found in the cytoplasmic and nuclear fractions of a baby hamster kidney cell line. They were separated by chromatography on DEAE-cellulose and partially purified by ammonium sulfate fractionation, DNA - cellulose and linear sucrose gradients. The cytoplasmic DNA polymerase exhibited an S-coefficient of 6.95 S in 0.15 M NaCl and its activity was highly sensitive to inhibition by N-ethylmaleimide and elevated temperatures, regardless of the presence of DNA template or other cofactors. It was stimulated by monovalent salts in the order of NH4 Cl greater than KCl greater than NaCl greater than CsCl greater than LiCl (inhibitory). The DNA polymerase extracted from nuclei sedimented with an S-value of 3.47 S, was resistant to inactivation by N-ethylmaleimide, and maximally stimulated by NaCl, while also being inhibited by LiCl. For optimal activity, both DNA polymerase activities required a divalent cation, with MgCl2 being more effective than MnCl2. Although the optimal pH values for the two enzyme activities differed slightly, glycine - NaOH buffer induced an alkaline shift of 1.5 pH units in the optimum of both enzymes. This was accompanied by an increase in the effectiveness of MnCl2 relative to MgCl2 for the cytoplasmic DNA polymerase.     

Induction of differentiation in cultured mouse neuroblastoma cells by N-methyl-N'-nitro-N-nitrosoguanidine.

N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), a potent mutagen and carcinogen, induces differentiation uniquely in N-18 mouse neuroblastoma cells, when compared with that of dibutyryl cyclic adenosine monophosphate. After treatment with 10 μM MNNG for only 2 h, RNA and protein synthesis are stimulated together with neurite formation, while DNA synthesis and growth of the cells are inhibited indefinitely. Induction of neurite formation by MNNG is irreversible, being inhibited by actinomycin D or cycloheximide but recovering after withdrawal of these inhibitors.     

Search for a DNA gyrase in mammalian mitochondria.

Incorporation of labeled deoxynucleoside triphosphates into mtDNA by isolated rat liver mitochondria has been shown previously to reflect DNA replication. We have used this system to seek evidence for a mtDNA gyrase. Coumermycin, novobiocin, nalidixic acid, and oxolinic acid are known to be inhibitors of Escherichia coli gyrase, to inhibit E. coli DNA replication, to abolish colicin E1 replication, and to depress the supercoiling of phage lambda DNA, the last two via inhibition of the DNA gyrase. Our results show that these agents inhibit [3H]dATP incorporation into bulk mtDNA at concentrations similar to those used for E. coli. Analysis by sucrose gradient sedimentation confirms the inhibition and shows further that the synthesis of the highly supercoiled form of mtDNA (i.e. 39 S DNA) is depressed relative to other mtDNA forms (i.e. 27 S DNA), suggesting an inhibition of the supercoiling process. Analysis of the DNA by CsCl/propidium diiodide centrifugation shows, in addition, that incubation with coumermycin results in the appearance of a mtDNA form shown to be relaxed mtDNA. The results are consistent with the occurrence of a mtDNA gyrase and its operation in mtDNA replication.     

DNA synthesis in permeabilized mouse L cells.

Mouse L cells are rendered permeable to nucleoside triphosphates by a cold shock with a near isotonic buffer. These cells retain their morphologic integrity and use exogenously supplied nucleotides and deoxynucleotides to synthesize RNA and DNA. The newly synthesized DNA is nuclear and is the product of semiconservative replication. Incorporation of deoxynucleotides into DNA by thymidine kinase-deficient cells were used to conform rigorously that the exogenously supplied deoxynucleotides were incorporated into DNA without intermediate processing through nucleosides. DNA synthesis requires the presence of Na+, ATP, all 4 deoxynucleotides, and Mg2+. The reaction is inhibited by N-ethylmaleimide, p-hydroxymercuribenzoate and actinomycin D. Hydroxy-urea and arabinosylcytosine do not inhibit the reaction whereas cytosine arabinoside triphosphate shows competitive inhibition with the deoxynucleotides. These findings indicate that the permeable cell system can be used for in situ evaluations of the replicative DNA polymerase using the endogenous DNA template.     

Synthesis of DNA in permeabilized cells of Kluyveromyces lactis.

Kluyveromyces lactis cells permeabilized with nystatin, though no longer viable, were able to incorporate 3H-dATP into DNA. Maximum rate of synthesis was obtained when all four deoxyribonucleoside triphosphates were present. For prolonged incorporation of 3H-dATP into DNA rATP or phosphoenolpyruvate were of absolute requirement. DNA synthesis was inhibited by p-chloromercuribenzoate, N-ethylmaleimide, nalidixate, ethidium bromide and distamycin A. The density of DNA synthesized in permeabilized cells grown on non-fermentable and fermentable carbon sources was analyzed on CsCl gradients in the presence or absence of distamycin A. The DNA synthesized by permeabilized cells previously grown on glycerol was essentially mitochondrial DNA; nuclear DNA (30% of total) was also synthesized by cells previously grown on glucose.     

The effect of photodynamic treatment with thiopyronine on yeast nDNA and mtDNA

Summary In order to ascertain the main target of the photodynamic effect with the sensitizer thiopyronine (TP) and its interference with cellular DNA, the uptake of TP into the yeastSaccharomyces cerevisiae and the distribution of the dye within the cells were studied. After fractionation of the cellular components, about 1% of the TP was found to be bound to the nucleus in anaerobically grown yeast cells; in aerobically grown cells, about 7% could be detected at the mitochondria.After careful isolation of the DNA from the organelles, only 0.024% of the dye once taken up by the cells was detectable at nuclear DNA, whereas 0.652% was bound to mitochondrial DNA. A calculation of the number of TP-molecules bound per nucleotides revealed a ration of one molecule TP per about 6,000 nucleotides of nuclear DNA and one molecule TP per 53 nucleotides of mitochondrial DNA. In vitro, the maximal binding capacity was estimated to be about one molecule TP per nucleotide.The induction of single strand breaks in the DNA after photodynamic treatment in vivo and in vitro was investigated by comparing the sedimentation of nDNA and mtDNA through alkaline sucrose-gradients. No differences in the sedimentation profiles of nDNA after photodynamic treatment of the cells in vivo as compared to the untreated control could be observed. In contrast, the sedimentation coefficient of mtDNA was significally decreased after photodynamic treatment, indicating the induction of single strand breaks in vivo only in mtDNA and not in nDNA.     

Synthesis of high molecular weight DNA strands during S phase

The organization of the mammalian S phase was studied in synchronized mouse embryo cells in terms of the spatial relationship between replication units whose synthesis is initiated at different times in S phase and the rate of assimilation of replication units into high molecular weight DNA strands.The formation of high molecular weight nascent DNA strands several replication units in length was analyzed by velocity sedimentation in alkaline sucrose gradients and by isopycnic centrifugation in alkaline Cs₂SO₄/CsCl gradients. Differential labeling with an isotopic and a density label shows that replication units synthesized at different stages of the S phase are not found within the same high molecular weight polynucleotide strand. It is thus concluded that replication units duplicated at different stages of the S phase are spatially organized in clusters along the mammalian genome.The rate of formation of high molecular weight nascent DNA strands is at least 4 to 8 times slower than that predicted from the spatial organization of replication units and the rate of chain growth within replication units. It is concluded that the process of joining of the completed nascent strands of adjacent replication units plays a major role in the rate of completion of high molecular weight strands.     

The inhibition of mitochondrial DNA replication in vitro by the metabolites of benzene, hydroquinone and p-benzoquinone

Rat liver mitochondria incubated with the metabolites of benzene, p-benzoquinone or 1,2,4-benzenetriol, showed a dose-dependent inhibition of [3H]dTTP incorporation into mtDNA with median inhibitory concentrations of 1 mM for each compound. Benzene and the metabolites phenol, catechol and hydroquinone did not inhibit at concentrations up to 10 mM. Similarly, incubation of p-benzoquinone or hydroquinone with rabbit bone marrow mitochondria showed a dose-dependent inhibition of mtDNA synthesis with 50% inhibition at 1 mM and 10 mM, respectively. That these metabolites inhibit mitochondrial replication was evidenced by the fact that [3H]dTTP incorporation into characteristic 38S, 27S and 7S mitochondrial replication intermediates was decreased by the quinones, as analyzed on 5-20% neutral sucrose velocity gradients. p-Benzoquinone, hydroquinone and 1,2,4-benzenetriol inhibited the activity of partially purified rat liver mtDNA polymerase gamma using either activated calf thymus DNA or poly(rA) X p(dT)12-18 as primer/template, with 50% inhibitory concentrations of 25 microM, 25 microM and 180 microM, respectively. Preincubation of the metabolites with polymerase gamma or primer/template, followed by removal of the unreacted metabolite by gel filtration, indicated that inhibition resulted from interaction of the metabolites with the enzyme, rather than with the template. Binding appeared to involve a sulfhydryl residue on the enzyme since the binding of [14C]hydroquinone was prevented by N-ethylmaleimide. The ability of hydroquinone or p-benzoquinone to inhibit binding of [14C]hydroquinone to the enzyme suggests that the compounds bind to a common site or are converted to a common intermediate. Inhibition of, or changes in, replication in mitochondria of bone marrow cells by hydroquinone and p-benzoquinone may explain the changes in the mitochondrial genome observed in marrow stem cells in acute myelogenous leukemia and may suggest a mechanism for benzene leukemogenesis.     

Repair response of Escherichia coli to hydrogen peroxide DNA damage.

The repair response of Escherichia coli to hydrogen peroxide-induced DNA damage was investigated in intact and toluene-treated cells. Cellular DNA was cleaved after treatment by hydrogen peroxide as analyzed by alkaline sucrose sedimentation. The incision step did not require ATP or magnesium and was not inhibited by N-ethylmaleimide (NEM). An ATP-independent, magnesium-dependent incorporation of nucleotides was seen after the exposure of cells to hydrogen peroxide. This DNA repair synthesis was not inhibited by the addition of NEM or dithiothreitol. In dnaB(Ts) strain CRT266, which is thermolabile for DNA replication, normal levels of DNA synthesis were found at the restrictive temperature (43 degrees C), showing that DNA replication was not necessary for this DNA synthesis. Density gradient analysis also indicated that hydrogen peroxide inhibited DNA replication and stimulated repair synthesis. The subsequent reformation step required magnesium, did not require ATP, and was not inhibited by NEM, in agreement with the synthesis requirements. This suggests that DNA polymerase I was involved in the repair step. Furthermore, a strain defective in DNA polymerase I was unable to reform its DNA after peroxide treatment. Chemical cleavage of the DNA was shown by incision of supercoiled DNA with hydrogen peroxide in the presence of a low concentration of ferric chloride. These findings suggest that hydrogen peroxide directly incises DNA, causing damage which is repaired by an incision repair pathway that requires DNA polymerase I.     

In vitro DNA and RNA synthesis by human platelets.

In vitro incorporation of [Me-3H] thymidine and [5-3H] uridine into human platelets was demonstrated. Thymidine incorporation was inhibited by three specific inhibitors of DNA synthesis: hydroxyurea, cytosine arabinoside and daunomycin. The effect was dose-dependent. Uridine uptake by platelets was found to be inhibited by specific inhibitors of RNA synthesis such as actinomycin D, rifampicin and vincristine, the effect of actinomycin D being dose dependent. The drug also led to a time-dependent inhibition of protein synthesis when preincubated with platelets. The platelet RNA profile on polyacrylamide gel was demonstrated to be similar to that of embryonic mouse erythroblast RNA. Synthesis of all three fractions, 28 S, 18 S and 4 S, was inhibited by actinomycin D. These findings show that human platelets are capable of DNA and RNA synthesis, and that these activities play a role in controlling protein synthesis in these cells. Detectable amounts of DNA have been found in whole human platelets, and in isolated mitochondria derived from these cells. Isolated platelet mitochondria incorporated [3H] thymidine and [3H] uridine into their macromolecules. These activities were inhibited by daunomycin and by both rifampicin and actinomycin D, respectively. These results support the assumption that DNA and RNA synthesis found in intact cell preparations takes place most probably in platelet mitochondria.     

Forms of Deoxyribonucleic Acid Produced by Virions of the Ribonucleic Acid Tumor Viruses

The in vitro product of mouse leukemia virus deoxyribonucleic acid (DNA) polymerase can be separated into two fractions by sedimentation in sucrose gradients. These two fractions were analyzed for their content of single-stranded DNA, double-stranded DNA, and DNA-ribonucleic acid (RNA) hybrid by (i) digestion with enzymes of known specificity and (ii) equilibrium centrifugation in Cs(2)SO(4) gradients. The major fraction early in the reaction contained equal amounts of single-stranded DNA and DNA-RNA hybrid and little double-stranded DNA. The major fraction after extensive synthesis contained equal amounts of single-and double-stranded DNA and little hybrid. In the presence of actinomycin D, the predominant product was single-stranded DNA. To account for these various forms of DNA, we postulate the following model: the first DNA synthesis occurs in a replicative complex containing growing DNA molecules attached to an RNA molecule. Each DNA molecule is displaced as single-stranded DNA by the synthesis of the following DNA strand, and the single-stranded DNA is copied to form double-stranded DNA either before or after release of the single strand from the RNA. Actinomycin blocks this conversion of single-to double-stranded DNA.     

DNA synthesis in isolated mitochondrial nucleoids from plasmodia ofPhysarum polycephalum

Summary A mitochondrion contains multiple copies of mitochondrial DNA (mtDNA) in the mitochondrial nucleoid (mt-nucleoid, synonym for mitochondrial nuclei). Replicaton of mtDNA in the mtnucleoids appears to be regulated within groups of adjacent mtDNA molecules, known as mitochondrial replicon clusters (MRCs). In this study, we isolated structurally intact mt-nucleoids from the plasmodia ofPhysarum polycephalum and characterized DNA synthesis in the isolated mt-nucleoids. The mt-nucleoids were isolated by dissolving the membranes of highly purified mitochondria with 0.5% Nonidet P-40. The structural integrity of the isolated mt-nucleoid was determined by observing the rod shape of the mt-nucleoid and the structure of the MRC. The isolated mt-nucleoids required four deoxyribonucleoside triphosphates and MgCl₂ for DNA synthesis. The DNA synthesis was resistant to aphidicolin and showed only low sensitivity to N-ethylmaleimide and to ddTTP, suggesting that the DNA synthesis is catalyzed by plant-type mitochondrial DNA polymerase. The capacity for DNA synthesis in the isolated mt-nucleoids was similar to that in the isolated mitochondria, despite removal of most of the mitochondrial matrix and membrane. Furthermore, visualization of sites of DNA synthesis in vitro revealed that DNA synthesis in the isolated mt-nucleoids occurred in each MRC. These results suggest that the isolated mt-nucleoids are capable of efficient and systematic DNA synthesis in vitro. Therefore, the use of isolated mt-nucleoids should permit in vitro characterization of the molecular mechanism of mtDNA replication in the MRC.Abbreviations BrdU 5-bromodeoxyuridine - BrdUTP 5-bromo-deoxyuridine triphosphate - DAPI 4,6-diamidino-2-phenylindole - dNTP deoxyribonucleoside triphosphate - ddCTP dideoxycytidine triphosphate - NEM N-ethylmaleimide - MRC mitochondrial replicon cluster; mt mitochondrial - NP-40 Nonidet P-40 - PBS phosphatebuffered saline - PMSF phenylmethanesulfonyl fluoride - rNTP ribonucleoside triphosphate - VIMPCS video-intensified microscope photon-counting system     

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.