Summary
Isolation and characterization of Chinese hamster ovary cell mutants resistant to different DNA polymerase ase inhibitors (aphidicolin, ara-A and ara-C) have been described. A particular mutant (JK3-1-2A) characterized in detail was found to grow and synthesize DNA in medium containing an amount of aphidicolin tenfold greater than that which completely inhibited the growth and the DNA synthesis of the wild-type cells. An almost twofold increase in the specific activity of the DNA polymerase α was seen in this mutant. The mutant DNA polymerase showed altered aphidicolin inhibition kinetics of dCMP incorporation; the apparent K m for dCTP and the apparent K i for aphidicolin were increased in the mutant. These alterations in the kinetic parameters were, however, abolished upon further purification of the enzyme. Ara-CTP was found to act as a competitive inhibitor of the dCMP incorporation by both the wild type and mutant enzymes. In contrast, the effect of aphidicolin on dCMP incorporation was either competitive (wild-type enzymes) or noncompetitive (mutant enzyme). The data presented showed that the sites of action for aphidicolin and ara-CTP were distinct; likewise the dCTP binding site appeared to be separate from other dNTP(s) binding sites. The drug resistance of the mutant was inherited as a dominant trait.
Similar content being viewed by others
Abbreviations
- ara-A:
-
9-β-d-arabinofuranosyl adenine
- ara-C:
-
1-β-d-arabinofuranosyl cytosine
- aph:
-
aphidicolin
References
Alberts BM, Barry J, Bedings P, Burke RC, Hubner U, Liu CC, Sheridan R (1980) Studies on the replication mechanism with the T4 bacteriphage in vitro system. In: Alberts BM, Fox F (eds) ICN-UCLA symposium on molecular, cellular biology, vol XIX. Academic Press, New York, pp 449–473
Aposhian HV, Kornberg A (1962) Enzymatic synthesis of deoxyribonucleic acid: The polymerase formed after T2 bacteriophage infection of E. coli: A new enzyme. J Biol Chem 237:519–525
Ayusawa D, Iwata K, Kozu T, Ikegami S, Seno T (1979) Increase in dATP pool in aphidicolin-resistant mutants of mouse FM3A cells. Biochem Biophys Res Commun 91:946–954
Ayusawa D, Iwata K, Seno T (1981) Alteration of ribonucleotide reductase in aphidicolin resistant mutant of mouse cell with associated resistance to arabinosyladenine and arabinosylcytosine. Somat Cell Genet 7:27–42
Banks GR, Boezi JA, Lehman IR (1979) A high molecular weight DNA pol from Drosophila melanogaster. J Biol Chem 254:9886–1892
Baril E, Baril B, Elford H, Luftig RB (1973) DNA polymerases and a possible multi-enzyme complex for DNA biosynthesis in Eukaryotes. In: Kober AR, Kohiyama M (eds) Mechanism and regulation of DNA replication. Plenum Publishing Corp, New York, pp 275–291
Baril E, Bonin P, Burnstein D, Mara K, Zamecnik P, Baril E (1983) Resolution of Ap4A binding proteins in Hela cells. Proc Natl Acad Sci 80:4931–4935
Chang C-C, Boezi JA, Warren ST, Sabourin CLK, Liu PK, Glatzer L, Trosko JE (1981) Isolation and characterization of a UV sensitive hypermutable aphidicolin resistant Chinese hamster cell. Somat Cell Genet 7:235–253
Cohen SS (1977) The mechanism of the lethal action of ara-C and ara-A. Cancer 40:509–518
Cozzarelli NR (1977) The mechanisms of action of inhibitors of DNA synthesis. Annu Rev Biochem 46:641–668
Davidson RL, Park GS (1976) Improved techniques for the induction of mammalian cell hybridization by polyethylene glycol. Somat Cell Genet 2:165–176
DeLucia P, Cairns J (1969) Isolation of an E. coli strain with a mutation affecting DNA polymerase. Nature 224:1164–1169
DeSaint-Vincent BR, Buttin G (1979) Studies on 1β-D arabinofuranosyl cytosine resistant mutants of Chinese hamster fibroblast. Somat Cell Genet 5:69–82
Fry M (1982) Eukaryotic DNA polymerases. In: Jacob ST (ed) Enzymes of DNA synthesis and processing. CRC Press, Boca Raton, Florida, pp 39–92
Huberman JA (1981) New view of the biochemistry of eukaryotic DNA replication revealed by aphidicolin, an unusual inhibitor of DNA pol α. Cell 23:647–648
Ikegami S, Taguchi T, Ohashi M, Oguro M, Nagano H, Mano Y (1978) Aphidicolin prevents mitotic cell division by interfering with the activity of DNA pol α. Nature (London) 275:458–460
Kammen HO (1966) A rapid assay for thymidylate synthetase. Anal Biochem 17:553–556
Kornberg A (1980) DNA replication. W.H. Freeman & Co, San Francisco
Krokan H, Schaffer P, DePamphilis ML (1979) Involvement of DNA polymerase α and γ in the replication of cellular and viral deoxyribonucleic acid. Biochemistry 18:4431–4443
Lamothe P, Baril B, Chi A, Lee L, Baril E (1981) Accessory proteins for DNA polymerase α activity with single stranded DNA templates. Proc Natl Acad Sci USA 78:4723–4727
Lewis W, Wright J (1974) Altered ribonucleotide reductase activity in mammalian tissue culture cells resistant to hydroxyurea. Biochem Biophys Res Commun 71:128–135
Lindberg U, Skoog L (1970) A method for the determination of dATP and dTTP in picomole amounts. Anal Biochem 34:152–160
Liu PK, Chang CC, Torsko JE, Dube DK, Martin GM, Loeb LA (1983) Mammalian mutator mutant with an aphidicolin-resistant DNA polymerase α. Proc Natl Acad Sci 80:797–801
Lowry DH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275
Migeon BR, Smith SW, Leddy CL (1969) The nature of thymidine kinase in the human mouse hybrid cell. Biochem Genet 3:583–590
Mishra NC (1983) DNA polymerase mutants of Neurospora. Proceedings of the 15 International Genetics Congress, part II. Oxford and IBH Press, New Delhi, pp 500
Mishra NC (1985) DNA repair defective mutants of Neurospora. In: Proceedings of the symposium on applied and basic mutagenesis. Oxford and IBH Press, New Delhi (in press)
Mishra NC, Hinnant K, Cason E (1985) New ara-C resistant mutants of Chinese hamster ovary cells. Genet Res (in press)
Nishimura M, Yasuda H, Ikegami S, Ohashi M, Yamada M (1979) Aphidicolin resistant mutant of which DNA pol α is induced by this drug. Biochem Biophys Res Commun 91:939–945
North TW, Bestwick RK, Mathews CK (1980) Detection of activities that interfere with the enzymatic assay of deoxyribonucleoside 5′-triphosphates. J Biol Chem 255:6640–6645
Oguro M, Suzuiki-Hori C, Nagano H, Mano Y, Ikegami S (1979) The mode of inhibitory action by aphidicolin on eukaryotic DNA pol α. Eur J Biochem 97:603–607
Puck TT (1972) The mammalian cell as microorganism. Holden Day Inc, San Francisco, pp 1–230
Rapport E, Zamecnik PC (1976) Presence of diadenosine S1-S111-P1-P4 tetraphosphate (Ap4A) in mammalian cells in levels varying widely with a proliferative activity — polytypic activator. Proc Natl Acad Sci USA 73:3984–3989
Rashbaum SA, Cozzarelli NR (1976) Mechanism of DNA synthesis inhibition by arabinosyl cytosine and arabinosyl adenine. Nature 264:679–680
Reddy GPV, Pardee AB (1982) Coupled ribonucleotide diphosphate reduction channeling and incorporation into DNA of mammalian cells. J Biol Chem 257:12526–12531
Sabourin CLK, Bates PF, Glatzer L, Chang C-C, Trosko JE, Boezi JA (1981) Selection of aphidicolin resistant CHO cells with altered levels of ribonucleotide reductase. Somat Cell Genet 7:255–268
Schimke RT, Kaufman RJ, Alt FW, Kellems RF (1978) Gene amplification and drug resistance in cultured murine cells. Science 202:1051–1055
Skoog L (1970) An enzymatic method for the determination of dCTP and dGTP in picomole amounts. Eur J Biochem 17:202–208
Smith DB, Mishra NC (1985) A Chinese hamster ovary cell mutant resistant to aphidicolin. Cell biol (in press)
Sugino A, Nakayama K (1980) DNA polymerase α mutants from a Drosophila melanogaster cell line. Proc Natl Acad Sci USA 77:7049–7053
Sugino A, Kojo H, Greenberg BD, Brown PO, Kim KC (1981) In vitro replication of a yeast 2 μm plasmid DNA. In: Fox CF (ed) The initiation of DNA replication — ICN-UCLA symposium. Academic Press. New York, pp 529–553
Ullman B, Cliff SM, Gudas LJ, Levison BB, Wormsted MA, Martin DW (1980) Alteration in deoxyribonucleotide metabolism in cultured cells with ribonucleotide reductase activities refractory to feedback inhibition by 2′deoxyadenosine triphosphate. J Biol Chem 255:8308–8314
Vishwanatha JK, Mishra NC (1983) Aphidicolin resistant CHO cells with altered DNA polymerase α. Proceedings of the XV International Genetics Congress, part I. Oxford and IBH Press, New Delhi, p 503
Vishwanatha JK, Mishra NC (1985) Chinese hamster ovary cell mutants resistant to DNA polymerase inhibitors. II. Segregational analysis and DNA transfection of aph r gene. Mol Gen Genet 200:401–406
Weinberg G, Ullman B, Martin DW Jr (1981) Mutator phenotypes in mammalian cell mutants with distinct biochemical defects and abnormal dNTP pools. Proc Natl Acad Sci USA 78:2447–2451
Author information
Authors and Affiliations
Additional information
Communicated by K. Illmensee
Rights and permissions
About this article
Cite this article
Vishwanatha, J.K., Mishra, N.C. Chinese hamster ovary cell mutants resistant to DNA polymerase inhibitors. Molec. Gen. Genet. 200, 393–400 (1985). https://doi.org/10.1007/BF00425722
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00425722