DNA Polymerase A-Primase Complexes from Carcinogen-Treated Chinese Hamster Ovary Cells1

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(CANCER RESEARCH 50, 6894-6901, November I. 1990] DNA Polymerase a-Primase Complexes from Carcinogen-treated Chinese Hamster Ovary Cells1

Angelika Brucker, Lawrence A. Loeb, and Heinz Walter Thielmann2

German Cancer Research Center, Institute of Biochemistry', Im Keuenheinier Feld 280, 6900 Heidelberg, Federal Republic' of Germany fA. B., //. W. T.J, ana The Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology SM-30, University of Washington, Seattle, Washington 98195 [L. A. L.Â¡

ABSTRACT or as a cryptic activity (9), although indirect evidence now has been presented that exonucleolytic proofreading occurs during In order to investigate whether carcinogens induce alterations of the DNA replication (10). Additional to exonucleolytic proofread DNA polymerase a-primase complex we compared the physicochemical ing, fidelity with DNA polymerase Â«,the major replicating and catalytic properties and the fidelity of DNA synthesis of DNA polymerase a-primase complexes from carcinogen-treated and untreated enzyme is controlled mainly by selection of the complementary Chinese hamster ovary cells. Complexes were purified by ion exchange base. It has been frequently postulated that alterations in DNA or by immunoaffinity Chromatograph) and both DNA-polymerizing ac polymerases by chemical carcinogens might promote misincor- tivities and those of ancillary enzymes, such as RNA primase and poration du ring the initiation of the carcinogenic process or exonuclease, were examined. Further characterization of the complexes during tumor progression (11, 12). included determination of the relative molecular masses, sedimentation In fact, a cytoplasmic DNA polymerase Â«witha lower fidelity coefficients, and diffusion coefficients, and measurements of the Kmsfor than that of normal rat liver was isolated from livers of rats fed deoxynucleotide triphosphates and DNA templates, which were identical /V-2-fluorenylacetamide (13). Although the mechanism by for the preparations from both carcinogen-treated and untreated cells. The fidelity of DNA polymerase a-primase complexes measured by the which mutations are introduced in mammalian cells is still not X174am3reversion assay was also similar in carcinogen-treated and known in detail, this result indicates that mutagenesis might be untreated cells. Thus, a carcinogen-mediated induction of a DNA polym controlled at the level of DNA polymerase a. Therefore, the erase a-primase complex with low fidelity was not observed within the aim of our study was to elucidate whether or not an error-prone detection limits of the 0X174 assay. DNA polymerase a-primase complex is involved in mutagene RNA primase was found to be an ancillary enzyme activity of the DNA sis. polymerase a from both carcinogen-treated and untreated cells; however, In E. coli an error-prone pathway for mutagenesis has been the RNA primase:DNA polymerase a activity ratio was significantly studied extensively. Mutations caused by UV light, ionizing higher in DNA polymerase a-primase complexes from carcinogen-treated radiation, and many carcinogens arise as the result of an induc- cells. These complexes also exhibited an at least 3 times greater velocity ible error-prone repair pathway which is part of the SOS of synthesis with supercoiled or unprimed single-stranded DNAs as templates. Since the binding sites of DNA polymerase a-primase com response (14, 15). This response is controlled by the recA. and plexes for deoxynucleotide triphosphates and DNA templates were shown lexA genes (for review, see Ref. 16). The precise mechanism of to be identical before and after treatment of cells with carcinogens (i.e., SOS-induced mutagenesis is unknown, but it has been proposed identical A,,,values for different DNA templates and A values for specific that SOS induction results in a transient decrease in the fidelity inhibitors), the increased synthesis catalyzed by the DNA polymerase a- of DNA replication, which in turn facilitates DNA synthesis primase complex from carcinogen-treated cells might be due to a carcino past template lesions (17, 18) and thus leads to both targeted gen-induced alteration of an accessory protein of the complex. and untargeted mutagenesis (19-22). There is evidence that DNA polymerase III is involved in the mutation fixation pro INTRODUCTION cess during UV mutagenesis (23, 24). A model for SOS muta genesis in E. coli was proposed recently (25, 26), in which it Normally, DNA replication is an extremely accurate process; was assumed that irons-lesion synthesis occurs in two steps. error frequencies are ~10~9-10~"/base replicated (1). This high The first step (misincorporation) is believed to be mediated by fidelity is thought to occur, at least in Escherichia coli, through DNA polymerase III (27) and results in the insertion of an a sequence of mechanisms (2-4): (a) selection of the comple incorrect base opposite a pyrimidine dimer. The second step mentary base by the replicative DNA polymerase;3 (b) exonu- (bypass) would require high levels of the DNA-binding proteins cleolytic 3'-5' editing of a noncomplementary base at the UmuC and UmuD which allow polymerase III to replicate past growing point; and (c) postreplicative mismatch repair. the blocking lesion (28). In eukaryotes, however, the 3'-5'-exonuclease did not appear Many of the distinctive features of the prokaryotic SOS to be a consistent constituent of isolated polymerase Â«for a system are absent in eukaryotic cells. Several lines of evidence long time, inasmuch as it was found only exceptionally (5-8) indicate, however, that mammalian cells treated with chemical Received 4/3/90; accepted 7/13/90. carcinogens or UV light induce several emergency functions The costs of publication of this article were defrayed in part by the payment analogous to those in prokaryotes. These include: (a) virus of page charges. This article must therefore be hereby marked advertisement in reactivation (29, 30); (b) virus mutagenesis (31); and (c) gene accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This research was supported in part by an International Cancer Research amplification (32); in addition, synthesis of recombinational Technology Transfer (ICRETT) grant of the International Union Against Cancer and repair enzymes is enhanced (30, 33). Carcinogen-mediated (U1CC). Geneva, to A. B.. and by NIH Grant R35-CA39903 to L. A. L. 3To whom requests for reprints should be addressed. DNA amplification in hamster cells is inhibited by aphidicolin 1The abbreviations used are: DNA polymerase, deoxynucleoside triphos- and protease inhibitors but not by cycloheximide (34). There phate:DNA deoxynucleotidyltransferase (DNA-directed) (EC 2.7.7.7); RNA pri fore, it was suggested that DNA polymerase Â«in carcinogen- mase, nucleoside triphosphate:RNA nucleotidyltransferase (DNA-directed) (EC 2.7.7.6); Me(NO)(NO2)Gdn. /V-methyl-yV'-nitro-A'-nitrosoguanidine; MeNOUr, treated cells catalyzed DNA amplification and was biochemi ,V-methyl-.V-nitrosourea:butylanilino-dATP.2-(/j-n-butylanilino)-2'-deoxyadeno- sine 5'-triphosphate: bulylphenyl-dGTP. A'2-(/>-Â«-butylphenyl)-2'-deoxyguano- cally distinguishable (possibly posttranslationally modified) sine 5'-triphosphate: dNTP. deoxynucleoside triphosphate; dNMP, deoxynucleo from DNA polymerase Â«in untreated cells (34) and that DNA side monophusphate; AAV. adeno-associated virus. polymerase n was involved in this "SOS-like" response. 6894

In this study we compared DNA polymerase Â«-primasecom sence of mycoplasms was confirmed using the diamidinophenylimida- plexes from carcinogen-treated and untreated Chinese hamster zole assay (39). ovary cells with regard to the following characteristics: (a) Purification of DNA Polymerase a-Primase Complexes physicochemical properties, such as sedimentation and diffu sion coefficients and molecular masses; (b) interaction with Buffers. All buffers used for preparation of cell-free extracts and DNA and dNTPs monitored by determining kinetic parameters purification of DNA polymerase a-primase complexes contained 2 mivi such as Michaelis-Menten and inhibition constants; (c) fidelity dithiothreitol, 10 niM sodium bisulfite, 0.1 mM phenylmethylsulfonyl of DNA synthesis, determined by means of the <Â£X174am3 fluoride, 1 Mg/ml pepstatin A, and 1 Mg/ml leupeptin. Protocol of Treatment with Carcinogen. Cells were seeded into 625- reversion assay (35); and (d) associated activities of DNA cm2 dishes at a density of approximately 3 x 10' cells/cm2. Twenty- polymerase a. We used Me(NO)(NO2)Gdn and MeNOUr, both four h later, a part of the cells was treated with the carcinogens of which have been reported to induce SOS mutagenesis in Me(NO)(NO2)Gdn or MeNOUr, freshly dissolved in distilled dimethyl prokaryotes (16) and DNA amplification in mammalian cells sulfoxide (the final concentration of the solvent did not exceed 0.1%). (34, 36). The latter is suggested to represent one of the SOS- Control cells were treated with 0.1% dimethyl sulfoxide. The carcino like functions in animal cells (32). gens remained in the medium for 48 h. Cells were harvested in late exponential phase after 5 doublings (approximately 3 x IO5cells/cm2) by scraping them from the surface with a rubber policeman and collected MATERIALS AND METHODS by centrifugation. Cell pellets were washed three times in 10 mM phosphate-buffered saline, pH 7.4 (Serva), and stored at -8Â°C. Chemicals Unlabeled deoxynucleoside 5'-triphosphates, nucleoside 5'-triphos- Purification of DNA Polymerase a phates, 2',3'-dideoxythymidine 5'-triphosphate, and aphidicolin were Conventional Chromatography. A procedure of minimal purification purchased from Boehringer Mannheim, Mannheim, Federal Republic of Germany. Labeled [3H]dTTP (specific activity, 80 Ci/mmol) was was performed in order to prevent disruption of concerted interactions among DNA-replicating proteins (40). Each preparation started with from New England Nuclear, Dreieich, Federal Republic of Germany, 2-5 x 10" cells. Protein was measured according to the method of and [Â«-32P]dTTP(specific activity, 800 Ci/mmol) was obtained from Amersham-Buchler, Braunschweig, Federal Republic of Germany. N- Bradford (41) and determined by absorbance at 595 nm. Enzyme Ethylmaleimide and Â«-amanitin were bought from Sigma, Munich, activity was measured. Active fractions were combined (fraction IV), concentrated (fraction IVa) by means of a Centricon microconcentrator Federal Republic of Germany. Butylanilino-dATP and butylphenyl- (Amicon, Witten, Federal Republic of Germany), and stored at â€”70Â°C dGTP were gifts from G. Wright, University of Massachusetts Medical in buffer (50 mM potassium phosphate, (pH 7.5), 150 mM KC1, 50% School, Worcester, MA. Me(NO)(NO2)Gdn and MeNOUr were pur glycerol). chased from Sigma. The protease inhibitors pepstatin A, leupeptin, and Immunoaffinity Purification. DNA polymerases a were extensively phenylmethylsulfonyl fluoride and other chemicals were from Sigma. purified by immunoaffinity using the monoclonal antibody SJK 237- DNA Templates 71 (42) which is directed against DNA polymerase a (43). Fractions containing DNA polymerase a were combined (fraction IV-1), dialyzed Single-stranded, circular bacteriophage M13mp9 DNA was pur overnight against 2 liters of buffer (50 mM Tris-HCl, (pH 7.5), 10% chased from Boehringer Mannheim. Calf thymus DNA was from glycerol), concentrated (fraction IV-2), and stored as described above. Sigma. Activated calf thymus DNA was prepared using DNase I (37). MH1, a pMARS3 harboring E. coli strain, was a gift from Professor Enzyme Assays Grummt, Institute of Microbiology, WÃ¼rzburg,Federal Republic of DNA Polymerase a. This was assayed as described in Ref. 40. Germany. The plasmid DNA was prepared by means of the alkaline Nucleotide incorporation was calculated by determination of acid- lysis method (38). P4 phage DNA was made available by Dr. M. insoluble radioactivity (44). KrÃ¶pelin. One unit of polymerase catalyzes the incorporation of 1 nmol nucleotide in 60 min at 37Â°C(45). Proteins RNA Primase. The activity was determined with the coupled RNA Trypsin was from Boehringer Mannheim; DNase I and RNase I were primase/DNA polymerase assay (6). Acid-insoluble radioactivity was from Sigma. Protein standards used as markers for nondenatured gel measured as described above. electrophoresis were from Pharmacia, Freiburg, Federal Republic of One unit of RNA primase catalyzes the incorporation of 1 nmol of Germany. Bovine serum albumin, aldolase, catalase, apoferritin, and labeled nucleotide in 60 min at 37Â°C(6). a2-macroglobulin, the proteins used as standards in gel permeation and 3'-5'-Exonuclease Activity. This was measured by the amount of 3'- sedimentation experiments, were purchased from Serva, Heidelberg, labeled nucleotides rendered acid-soluble (46). Federal Republic of Germany. Velocity Sedimentation in Glycerol Gradients Chromatographie Media Linear gradients (10-30%) in neutral glycerol were prepared (47). DEAE-cellulose DE23 was from Whatman, Maidstone, Kent, Eng DNA polymerase a-primase complex was mixed with catalase and land; Sephadex G-25 and SuperÃ³se 12 gel filtration columns and layered on top of the gradients. The sample and other marker proteins MonoS cation exchange columns were purchased from Pharmacia. [bovine serum albumin (4.6S), aldolase (7.4S), catalase (l 1.3S), and Â«2- Protein A-Sepharose medium was from Bio-Rad, Munich, Federal macroglobulin (18.IS)], each layered on separate gradients, were sedi- Republic of Germany. mented in a Beckman SW60TÃŒrotorat 2Â°Cfor 15.5 h at 45,000 rpm. Twenty fractions were collected from the bottom of the tubes. Each Cell Culture fraction was analyzed for DNA polymerase activity and protein content The Chinese hamster ovary cell line (CHO clone K,-BH4, American (41). Then the sedimentation coefficient of the polymerase was deter Type Culture Collection, Rockville, MD) was used as the enzyme mined (47). source. Mycoplasma-free cultures were cultivated as monolayers on 625-cm2 Petri dishes (Nunc, Wiesbaden, Federal Republic of Germany) Gel Permeation Analysis in Ham's F-12 medium, pH 7.3, supplemented with 5-10% fetal calf Gel filtration was performed with a Pharmacia SuperÃ³se 12 column serum, penicillin G (100 IU/ml), and streptomycin (100 Mg/ml). Ab previously equilibrated with elution buffer [20 mM Tris-HCl (pH 7.5), 6895

30 HIM KCI, 0.1 HIM EDTA, 2 mM dithiothreitol, 10 mM sodium treated cells in neutral glycerol gradients showed that the major bisulfite, 0.1 HIMphenylmethylsulfonyl fluoride, l pg/ml pepstatin A, activities sedimented with >18.1S and 13.OS, two minor peaks l /jg/ml leupeptin, 20% glucose, and 0.05 mg/ml bovine serum albu sedimented with 10.5S and 8.OS. DNA polymerase Â«-primase min]. The column was calibrated with bovine serum albumin, aldolase, catalase, and apoferritin, with diffusion coefficients of 5.9 x 10~7cm2/s, complex from control cells exhibited almost identical sedimen 4.3 x IO"7cm2/s, 4.1 x 1(T7 cm2/s, and 3.6 x 10~7cm2/s (48). tation coefficients (>18.1S, 12.8S, 10.2S, and 7.9S). The pat tern of these activity peaks was the same as in gel filtration DNA Synthesis in Nondenatured Activity Gels analysis. The relative molecular masses of the peaks were calculated (53) using the Svedberg formula Nondenatured 4-10% polyacrylamide slab gels in buffer A [375 mM Tris-HCl (pH 8.9), 1 mM EDTA, 0.01% Triton X-100, 0.034% am Mr.2oT = RTs/D(\ - \p) monium persulfate, and 0.062% Ar,/V,A'',A''-tetraniethylenediamine containing 0.2 mg/ml activated calf thymus DNA and 0.05 mg/ml where M, is the relative molecular mass, R the gas constant, T fibrinogen, were used (47, 49, 50). The samples were diluted in buffer the absolute temperature, v the partial specific volume [assumed B [50 mM Tris-HCl (pH 7.5), 40% sucrose, 30% glycerol, and 0.5% to be 0.74 ml/g (54)], and p the density of water at 20Â°C(0.998 bromophenol blue) and ethanethiol was added to a final concentration of 1%. The samples were warmed up to 37Â°Cfor 3 min before being g/cm'). DNA polymerase Â«-primasecomplex from carcinogen- loaded onto the gel. Separation of proteins was performed in buffer C treated cells was shown to be associated with four peaks having (25 HIMTris base, 192 mM glycine, 0.01% Triton X-100) at 100 mA relative molecular masses of >485,000, 330,000, 244,000, and and 15Â°C. 166,000, which were almost identical to those of DNA poly Processing of the gel and autoradiographic analysis were performed merase Â«-primase complex from control cells (>485,000, using the protocol of Khan and Brown (47). 324,000, 237,000, and 165,000). 4>X174am3Fidelity Assay Catalytic Properties of the DNA Polymerase a-Primase Com plexes The modified 0X174am3 fidelity system (51) was used. In particular, a synthetic 15-nucleotide oligomer was hybridized with single-stranded In order to characterize the catalytic core, the binding sites 0X174am3 viral DNA at a primentemplate molar ratio of 10:1. The 3'-terminus of the hybridized oligomer was located three nucleotides of fraction IVa for DNA templates and dNTPs were analyzed. The binding site for DNA templates was characterized by the away from the amber codon. Km values of DNA synthesis using various templates. The All assays contained 0.2 ^g of primed 0X174am3 DNA and 0.02- dNTP-binding sites were characterized" by the K, values for 0.2 unit of the indicated DNA polymerase a-primase complex. Nucleo- specific inhibitors, which interfere competitively. Table 2 dis tide incorporation was calculated by assaying an aliquot for acid- plays the A'mvalues for synthesis on activated calf thymus DNA insoluble radioactivity (44). E. coli spheroplasts were transfected with and single-stranded M13 DNA as templates. Both A"mvalues DNA copied by DNA polymerases and the reversion frequency was determined by the progeny phage method (52). Background reversion were found to be the same for both complexes. Concluding frequency was determined with primed 0X174am3 DNA which had from Kmvalues the binding sites of the complexes from carcino not been incubated with DNA polymerase (uncopied DNA). Experi gen-treated and untreated cells did not differ from each other. mental accuracy was expressed as the variation of reversion frequencies The DNA polymerase Â«-primase complexes tested were all of duplicates, which was usually less than 30%. sensitive to inhibition by aphidicolin, butylanilino-dATP, butylphenyl-dGTP, and Â¿Y-Ethylmaleimide(Table 3). Aphidicolin RESULTS is competitive with dCTP (56); butylanilino-dATP and butylphenyl-dGTP are structural analogues of the deoxynucleoside Purification and Physicochemical Properties of DNA Polymerase triphosphates dATP and dGTP (57); A'-Ethylmaleimide blocks a-Primase Complexes the sulfhydryl groups of the enzyme (58). Concluding from K, Two methods of purification of DNA polymerase Â«wereused values the binding sites of DNA polymerases Â«from treated in this study. The first involved conventional chromatography and untreated cells were identical for all deoxynucleoside tri and resulted in an only 18-fold enrichment in DNA polymerase phosphates. The K, values of the DNA polymerase Â«-primase Â«activity. This nonextensive purification was adequate to re complexes distinguished the latter from the DNA polymerases move endonuclease and exonuclease activity as well as DNA /3, 7, and o. It cannot be excluded, however, that under these polymerase ÃŸ.The second method used immunoaffinity chro Chromatographie conditions applied DNA polymerase Ã³copu- rified with the DNA polymerase Â«-primase complexes (59). matography and resulted in the extensive purification of DNA However, this is unlikely since we failed to detect any 3'-5'- polymerase a as judged by the lack of any detectable amounts of Coomassie blue-staining material in the most purified frac exonuclease activity (see below). Since DNA synthesis was not inhibited by 2',3'-dideoxy- tion. thymidine 5'-triphosphate, a specific inhibitor of DNA poly Purification of DNA polymerase Â«-primase complexes by means of conventional chromatography (MonoS) was 11-18- merase ÃŸ(60), the latter enzyme was not a component of the DNA polymerase Â«-primasepreparations. fold (purification scheme; see Table 1). Complexes from both induced and uninduced cells eluted at 180 mivipotassium phos Fidelity of DNA Synthesis phate. The elution profiles showed no significant differences. Gel filtration analysis of fraction IVa from Me(NO)(NO:>) In the 0X174 fidelity assay, a primed single-stranded DNA Gdn-treated cells revealed four maxima of activity, two main template containing the amber! mutation is copied in vitro with activities with D2o.Â»<3.5 and 3.7 x IO"7 cnr/s and two minor a purified DNA polymerase (35). Incorporation of dATP, activities with D2o.*of 4.3 and 4.8 x 10~7cm2/s. Fraction IVa dCTP, or dGTP instead of dTTP at position 587 results in from control cells showed maxima of activity of identical dif reversion to wild type. After copying past the amber site, the fusion coefficients. partially double-stranded DNA molecules are transfected to Velocity sedimentation of fraction IVa from carcinogen- E. coli spheroplasts which complete replication and produce 6896

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1990 American Association for Cancer Research. DNA POLVMERASE Â«-PRIMASE COMPLEX Table I Conventional purification ofl).\A polymerase n-primase complexes from Me(\O)(i\Oi)Gtin- and .\le\Ol 'r-treated anil untreated Chinese hamster ovary cells One unit is the amount of enzyme which catalyzes the incorporation of 1 nmol of ('HjdTMP into acid-insoluble DNA in 60 min at 37"C. Protein amount and activity are referred to 1 x 10*cells. protein activity activity ield of aclivitv PurificationUntreated (mg)4.43.6ÃŽ.O0.13.83.42.70.074.23.32.80.07Total(units)19.420.517.84.812.313.511.64.014.013.511.92.5Specific(units/mgprotein)4.45.75.948.93.24.04.357.13.34.14.235.0V.(%)10010692251001099433100968418 cellsCell-free extractSephadex G-25DEAE-celluloseMonoS IV'a)Me(NO)(NO2Gdn-treated(fraction

cellsCell-free extractSephadex G-25DEAE-celluloseMonoS IV'a)MeNOUr-treated(fraction

cellsCell-free extractSephadex G-25DEAE-celluloseMonoS

(fraction IVa)Total

Table 2 DKA synthesis using various Di\A templates: A'mvalues Increasing amounts of DNA (activated and single-stranded) were incubated with fraction IVa (conventional chromatography) and DNA synthesis was measured as described in "Materials and Methods." The A'mvalues were read off the double reciprocal plot of l/v verviÂ»1/|.S"|values [55|. The slope of the line was calculated by applying linear regression analysis. Values are given as micromolar mononucleotide concentration and arc the means of at least three independent preparations. ND. not determined. Numbers in parentheses. SD. A',,,

Untreated Me(NO)(NO,)Gdn- MeNOUr-treated Template cells treated cells cells Gapped calf thymus DNA (DNA polymerase

Table 3 Inhibition ofI)\A polymerase a-primase complexes from carcinogen-treated and untreated Chinese hamster ovary cells: K, values Equal amounts of enzyme were preincubated for 20 min at O'C with increasing amounts of inhibitors. Then DNA template (activated calf thymus DNA) was added and DNA polymerase a activity was measured as described in "Materials and Methods." Values were calculated by linear regression analysis and are the means of at least three independent preparations. Inhibitor concentration which caused 50% inhibition (A'fvalue) of DNA synthesis was determined. For each study fraction IVa was used as a source of DNA polymerase Â»-primasecomplex. ND, not determined. Numbers in parentheses. SD. A',OIM)

InhibitorAphidicolin" cells"1.3(0.2) cells"1.1 cells1.5(0.2) (0.2) 14.1 x IO'3 10.7 x IO"3 Butylanilino-dATPButylphenyl-dCTP"A'-EthylmaleimideÂ° NDN (2.1 x IO-3) (1.6X IO"') 12.5 x IO'3 ll.9Â± IO"3 D900(100) (1.5 x IO'3) (1.4 x IO"3) 1200(200) 1300(200) 2'.3'-Dideoxvth\midine 5'-triphosphateUntreated 380 (60)Me(NO)(NO2)Gdn-treated 370 (90)MeNOUr-treated 320 (80) Â°Done with both fraction IVa and fraction IV-2. progeny phage. Reversion frequency is monitored by plating by UV-induced lesions per DNA template as DNA polymerase the transfected spheroplasts on bacteria that are permissive or Â«-primasecomplex from untreated cells (data not shown). This nonpermissive for expression of the amber codon. Table 4 could mean that DNA polymerase Â«-primase complex from shows average titers, reversion frequencies and numbers of carcinogen-treated cells was not able to bypass these lesions dNMP molecules incorporated per 0X174 molecule for DNA more efficiently than the complex from untreated cells. polymerase Â«-primase complexes of fraction IVa and the en Ancillary Enzyme Activities zymes purified by immunoaffinity chromatography (fraction IV-2). Two independent preparations of 0X174am3 DNA were To investigate whether ancillary enzymes of the DNA poly used. Values for reversion frequencies are uncorrected for spon merase Â«-primase complexes are altered due to carcinogen taneous reversion frequency of X174am3(background). The treatment we tested the complexes from carcinogen-treated and concentration of one of the noncomplementary nucleotides, untreated cells for nucleases, especially the 3'-5'-exonuclease, dATP was 50-fold greater than the other three nucleotide and RNA primase. substrates. 3'-5'-Exonuclease. The 3'-5'-exonucleolytic proofreading Considering the accuracy of the assay, reversion frequencies activity of prokaryotic DNA polymerases is known to make a of synthesis on ^>X174am3 DNA polymerases Â«from carcino major contribution to the fidelity of DNA synthesis in vitro. gen-treated and untreated cells did not differ from each other, Therefore, we assayed the ability of the DNA polymerase a- irrespective of the method used for isolation of the respective primase complex (from treated and untreated cells) to digest enzyme. 3'-labeled nonrandomly associated polydeoxyadenylate-oligo- We also examined DNA synthesis on UV-treated single- deoxythymidylate templates to acid-soluble nucleotides. Nei stranded M13mp9 DNA. DNA polymerase Â«-primasecomplex ther a 3'-5'-exonuclease nor other nucleases were detected in from carcinogen-treated cells was inhibited to the same extent all DNA polymerase Â«-primasepreparations tested (results not 6897

Table 4 Fidelity ofD\A polymerase a-primase complexes from carcinogen- treated and untreated cells (fraction l\'a and IV-2) Synthesis on Various DNA Templates The number of independent experiments in Column 1 is given in parentheses; 0.2 unit fraction IVa and 0.02 unit fraction IV-2 were assayed, respectively. Two DNA synthesis catalyzed by fraction IVa from carcinogen- preparations of 0X174am3 DN A were used. The reversion frequency of uncopied treated and untreated cells was further analyzed using super- 0X174am3-DNA was 1.5 x IO"6(single experiments) or (3.4 Â±0.3) x 10~'(three coiled, linear double-stranded, and linear single-stranded DNAs or more independent experiments). Numbers in parentheses in column 3. SD. as templates. DNA synthesis on supercoiled DNA proceeded Phage titers Molecules dTMP Reversion incorporated/ only after initiation was performed. Therefore, a replication amherS Wild type frequency 0X174am3 system including all four rNTPs and an ATP-regenerating x 10"' x 10"! x 10~' DNA polymerase n molecule system keeping the ATP concentration constant was used in Conventionally purified DNA polymerase Â«-primasecomplex order to facilitate initiation. The templates used were: (a) the Control cells (6) 168 12.7 7.6(1.0) 14.9 plasmid pMARS3 which includes sequences of the genome of Control cells (1) 73 4.8 6.6 14.2 Me(NO)(NO,) Gdn- 237 18.9 7.9(2.1) 12.2 mice (62); and (b) the plasmids pAV2 and pA2Y 1which contain treated cells (3) sequences of an adeno-associated, helper virus-independent par- MeNOUr-treated cells 56 4.0 7.1 10.4 (I) vovirus (AAV2). Table 6 shows the velocities of DNA synthesis using various Immunoaffinity-purified DNA polymerase < templates catalyzed by the DNA polymerase Â«-primase com Control(3)Control cells (2.5)6.77.7(1.3)7.510.85.115.96.0 plex from carcinogen-treated cells. DNA synthesis which was (1)Me(NO)(NO;)cells Gdn-treated limited by the synthesis of the RNA primer was usually twice (3)Me(NO)(N02)cells as much as the control value. Gdn-treated cells (1)551802692005.112.020.715.09.2 In order to investigate the nature of this induced activity it was examined whether it acted on linear double-stranded and linear single-stranded DNA templates. With each of the tem shown). This result does not agree with the possibility that DNA polymerase o [which exhibits a 3'-5'-exonucleolytic ac plates DNA synthesis catalyzed by the complex from carcino gen-treated cells was more efficient by a factor of 2 than that tivity (61)] is part of the DNA polymerase Â«-primasecomplexes catalyzed by the complex from control cells (Table 6). purified. RNA Primase. The ability of the DNA polymerase Â«-primase complexes from carcinogen-treated and untreated cells to cat DISCUSSION alyze the synthesis of RNA primers on single-stranded DNA was examined by means of the coupled RNA primase/DNA Although the SOS model in E. coli has provided insight how polymerase assay. Synthesis on M13 single-stranded DNA did carcinogens might evoke mutations in bacteria, the precise not take place when rNTPs were absent, whereas in the presence mechanism for SOS mutagenesis still remains undefined. In principle, mutations can result from (a) UmuC-UmuD-induced of all four rNTPs together with dNTPs and DNA, the DNA changes in template conformation to compensate for distortion, polymerase a-primase complex catalyzed the incorporation of (b) inhibition of the 3'-5'-exonuclease (63), and/or (c) facili dNMPs on primer synthesis. Primer synthesis was insensitive tated binding of DNA polymerase III to the distorted primer- towards Â«-amanitin. Single fractions of the cation exchange template region, preventing dissociation and permitting multi column were assayed for both RNA primase and DNA poly ple replication attempts (28). Provided SOS-type mutagenesis merase Â«.The maxima of both enzymes coincided. The activity ratio determined for the complexes from Me(NO)(NO2)Gdn- in mammalian cells is based on similar mechanisms (29), it is and MeNOUr-treated cells, 0.086 and 0.081, respectively, was to be expected that alternatives b and c are accompanied by altered molecular properties of the DNA replication complex. higher than the ratio determined for the complex of control Therefore, DNA polymerase Â«-primasecomplexes from carci cells, 0.066. nogen-treated and untreated Chinese hamster ovary cells were Since the Km values for DNA synthesis on single-stranded compared with regard to their physicochemical and kinetic M13 DNA (see Table 2) were identical for all complexes tested, properties and fidelity of DNA synthesis. Attention was paid the increased activity of RNA primase was not caused by an not only to the core enzymes but also to accessory enzymic altered affinity of the binding sites of DNA polymerase ex activities. prÃmasecomplex to the DNA template. Using the same plot as Physicochemical and Kinetic Properties. When analyzed by- for the evaluation of Km values, Fmavvalues were determined sedimentation in neutral gradients and gel filtration DNA po (Table 5). Kmaxvalues for DNA synthesis on single-stranded lymerase Â«-primase complexes from carcinogen-treated and DNA catalyzed by DNA polymerase Â«-primasecomplexes from untreated cells exhibited four activity peaks. The complexes carcinogen-treated cells were higher than those determined with differed neither in diffusion nor in sedimentation values. Cal complexes from control cells. These findings confirm the in culation of the relative molecular masses from diffusion and creased ratio of RNA primase and DNA polymerase Â«. sedimentation constants (53) revealed identical molecular

Table 5 DNA synthesis using various DNA templates: rma, values Values were calculated by linear regression analysis and arc given in milliunits. Values are the means of at least three independent preparations. One unit of DNA polymerase <>or RNA primase is the amount which catalyzes the incorporation of 1 nmol [JH]dTMP into acid-insoluble DNA in 60 min at 37Â°C.Enzyme used was fraction IVa. ND. not determined. Numbers in parentheses. SD.

Template Untreated cells Me(NO)(NO;)Gdn-trealcd cells MeNOUr-treated cells Gapped calf thymus DNA (DNA 168.3(20.2) 153.2(16.9) ND polymerase

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1990 American Association for Cancer Research. DNA POLYMERASE Â«-PRIMASE COMPLEX Table 6 DNA synthesis catalyzed by DNA polymerase a-primase complexes those found for immunopurified DNA polymerases a from V- (fraction IVa) from carcinogen-treated and untreated cells using 79 hamster fibroblasts and TK-6 human lymphocytes (42). This various DNA templates The indicated fractions were incubated in 30 ^1 reaction buffer [40 mM 4-(2- result agrees with the fact that the binding of dNTPs and hydroxyethyl)-l-piperazineethanesulfonic acid, pH 8.0; 6 HIM MgCl2: 50 mM probably the specificity of base pairing during nucleotide selec KC1; 50 JIMEDTA; 1 mM dithiothreitol; 1 mM ATP; 40 /*Mconcentrations each of GTP, CTP, UTP, dATP. dGTP, and dCTP; 4 MM[3H]dTTP, 8.5% glycerol], tion was unaltered by carcinogen treatment. Although DNA containing an ATP-regenerating system (5 mM phosphoenolpyruvate, 1 unit polymerase o might have been present in the enzyme purified pyruvate kinase) for 5-30 min at 30Â°C.Incubation was stopped with 30 /il stop by conventional chromatography and could have accounted for buffer (10 mM Tris-HCI, pH 8.0, 10 mM EDTA, 0.45 M ammonium acetate, 0.1 mg/ml tRNA from yeast). DNA was precipitated, and acid-insoluble radio the high accuracy, it would not be a component of the immu activity was measured as mentioned in "Materials and Methods." The amount of nopurified polymerase. DNA template per assay applied (given in pmol nucleotides) was 1520 for pMARS3 and AAV2 single-stranded DNA and 760 pmol for all other DNA However, the 0X174am3 reversion assay is limited in that it templates indicated. Values for DNA synthesis are the mean of at least three monitors only single-base substitutions at this amber codon; independent preparations and are given as pmol dNMP incorporated per h. deletions, insertions, and frame-shift mutations which are also Numbers in parentheses, SD. caused by DNA polymerase a before and after SOS induction DNA synthesis (66) are not detectable in this system (67,68). Moreover, fidelity was measured by demonstrating untargeted mutagenesis. Keep Primer/templateSupercoiled cells26.8 treatedcells60.4(15.1) ing in mind these limitations and the accuracy of the method, DNA the carcinogens used did not induce a DNA polymerase ex pMARS3pAV2pA2YlUntreated (6.7) 20.8(5.2) 30.8 (7.7) prÃmasecomplex with lowered fidelity. 9.2 (2.3)Me(NO)(NO2)Gdn-20.8 (4.8) In addition, it should be noted that DNA polymerase a- primase complexes from carcinogen-treated and untreated cells Linear double-stranded DNA AAV2 5.6(1.4) 10.4(2.3) used UV-irradiated single-stranded DNA equally well for DNA synthesis. Linear single-stranded DNA AAV2 4.2(1.0) 9.0(2.1) Accessory Activities. The subunit composition of the DNA polymerase Â«-primasecomplex may influence not only fidelity but also the regular sequence and coordination of distinct steps weights (M, 165,000, 237,000, 324,000, and >485,000); each in DNA replication. Therefore, attention was focused on asso protein species was active when analyzed on nondenatured ciated enzyme activities (67), such as RNA primase and factors activity gels. The same pattern was found by Craig et al. (64, enabling the complex to catalyze DNA synthesis on double- 65). stranded DNA (possibly initiation factors). Since the complexes With regard to kinetic parameters, the DNA polymerase im catalyzed ribonucleotide-dependent but Â«-amanitin-indepen- primase complexes from carcinogen-treated and untreated cells dent synthesis on circular single-stranded DNA as a template, did not differ in K\ values for various inhibitors of the nucleo- RNA primase was not separated from DNA polymerase Â«by tide-binding site. Therefore, the binding of the nucleotides and, means of cation exchange and is therefore a tightly bound hence, the specificity of base pairing must be considered to be component of the complexes. The same observations were made identical for all DNA polymerase a-primase preparations for the purification of DNA polymerase Â«-primasecomplexes tested. The DNA binding site was characterized by means of from mouse cells, human KB cells, and calf thymus (69-71). Michaelis-Menten constants using two different DNA tem However, the ratio of RNA primase/DNA polymerase Â« plates. DNA polymerase a-primase complexes from carcino activity was higher in the complexes from carcinogen-treated gen-treated and untreated cells did not differ in their Kmvalues than in those from untreated cells. Correspondingly, Fmaxof for the templates tested. Therefore, the protocol for SOS in DNA synthesis (template: single-stranded DNA) catalyzed by duction [i.e., treatment with Me(NO)(NO2)Gdn or MeNOUr] the complexes from carcinogen-treated cells was 3-fold higher did not cause synthesis or posttranslational modification of than that catalyzed by the control complex. In addition, com DNA polymerases a yielding species with altered DNA binding plexes from carcinogen-treated cells catalyzed twice as much sites. DNA synthesis (templates: linear single-stranded, linear dou Summarizing these results it appears that both DNA poly ble-stranded DNA, and supercoiled DNA) as the complex from merase Â«-primasecomplexes possess an identical a-type, mul- control cells. It should be noted that nuclease activity was not tisubunit structure. Even so, it cannot be excluded that DNA detectable. Therefore, the increase in DNA synthesis was not polymerase 5 is a component of these complexes, as found for caused by unspecific nicking of DNA templates. Since the preparations purified from calf thymus (59). This seems, how velocities of DNA synthesis on activated calf thymus DNA ever, unlikely since a 3'-5'-exonuclease [which is part of DNA catalyzed by both complexes were of the same magnitude, one polymerase Ã³(61)] was not detectable in our fractions. must postulate an induced activity which seems to be part of Fidelity. Fidelity was determined by means of the 0X174am3 the complexes of carcinogen-treated cells and capable of en reversion assay (51). DNA polymerase a-primase complexes hancing (the initiation of) DNA synthesis on double-stranded from both treated and untreated cells catalyzed the synthesis DNA. past the amber site of X174am3.Furthermore, both prepara Since these studies were not done with Â¡mmunopurified DNA tions catalyzed the incorporation of an identical number of polymerases a, the results should be stated as preliminary. dNMPs per DNA molecule. Therefore, the calculation of the Nevertheless, an obvious explanation would be the existence of absolute number of copied $X174am3 molecules was unneces a complex-associated factor being increased in either amount sary. or activity. Such a factor could be RNA primase which catalyzes The reversion frequencies obtained with both DNA poly not only synthesis of Okazaki fragments but also the initiation merase a preparations were identical, regardless of whether of DNA replication at the origins of replication (72, 73). The cation or immunoaffinity chromatography was used for purifi biological significance of the elevated primase activity of carcino cation of the enzymes. They were of the same magnitude as gen-treated cells yet remains unclear. However, the chemical 6899

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1990 American Association for Cancer Research. DNA POLYMERASE Â«-PRIMASE COMPLEX carcinogen used might induce synthesis and/or posttransla- 4197, 1985. Bridges, B. A., and Woodgate. R. The two-step model of bacterial UV tional modification of a (initiation) factor which alters the mutagenesis. MutÃ¢t.Res.. ISO: 133-139, 1985. frequency of initiation of DNA replication. 27. Livneh, Z.. Shwartz. H.. Hevroni, D., Sharitt. O.. Tadmor. Y., and C'ohcn. O. Bypass and termination at lesions during in vitro DNA replication. Implications for SOS mutagenesis. In: R. E. Moses and W. C. Summers ACKNOWLEDGMENTS (eds.), DNA Replication and Mutagenesis. pp. 296-304. Washington. DC: American Society for Microbiology. 1988. We thank Dr. O. Yalkinoglu for providing pAV2, pA2Y and AAV- 28- Woodgate. R.. Rajagopalan, M.. Lu, C., and Echolson. H. 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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1990 American Association for Cancer Research. DNA Polymerase α-Primase Complexes from Carcinogen-treated Chinese Hamster Ovary Cells

Angelika Brucker, Lawrence A. Loeb and Heinz Walter Thielmann

Cancer Res 1990;50:6894-6901.

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