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

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DNA Polymerase A-Primase Complexes from Carcinogen-Treated Chinese Hamster Ovary Cells1 (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.¡ 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 «,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 «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 «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 «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-(/>-«-butylphenyl)-2'-deoxyguano- cally distinguishable (possibly posttranslationally modified) sine 5'-triphosphate: dNTP. deoxynucleoside triphosphate; dNMP, deoxynucleo from DNA polymerase «in untreated cells (34) and that DNA side monophusphate; AAV. adeno-associated virus. polymerase n was involved in this "SOS-like" response. 6894 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1990 American Association for Cancer Research. DNA POLYMERASE n-PRlMASE COMPLEX In this study we compared DNA polymerase «-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 <£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
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