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A simple and sensitive ribonucleotide reductase assay

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Abstract

Ribonucleotide reductase (RR) is a key regulatory enzyme in the DNA synthesis pathway and is the target of the cancer chemotherapeutic agent hydroxyurea. The study of RR is significantly hindered by the tedious and labor-intensive nature of enzymatic assay. In this report, we present a novel RR assay in which detection of the deoxyribonucleotides produced by RR occurs via coupling to the DNA polymerase reaction, and is enhanced by using RNase to degrade endogenous RNA. Cell extracts from various cell lines were treated with RNase and then reacted with ATP and radioactive ribonucleotide diphosphate as the substrate. Incorporation of the radioactive substrate [14C]CDP into DNA was linear over 30 min and was linear with the amount of extract, which provided RR activity. The reaction was inhibited by hydroxyurea and required Mg2+ and ATP, suggesting that the assay is specific to RR activity. While RR activities determined by our method and by a conventional method were comparable, this novel method proved to be simpler, faster, more sensitive and less expensive. In addition, assay of the RR activity for multiple samples can easily be performed simultaneously. It is superior to other RR assays in all aspects.

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References

  1. 1

    Albert DA, Nodzenski E, Yim G, Kowalski J. Effect of cyclic AMP on the cell cycle regulation of ribonucleotide reductase M2 subunit messenger RNA concentrations in wild-type and mutant S49 T lymphoma cells. J Cell Physiol 143:251–256;1990.

  2. 2

    Albert DA, Rozengurt E. Synergistic and coordinate subunits in Swiss 3T3 cells: Effect of multiple signal-transduction pathways. Proc Natl Acad Sci 89:1597–1601;1992.

  3. 3

    Ali MA, McWeeney D, Milosavljevic A, Jurka J, Jariwalla RJ. Enhanced malignant transformation induced by expression of a distinct protein domain of ribonucleotide reductase large subunit from herpes simplex virus type 2. Proc Natl Acad Sci USA 88:8257–8261;1991.

  4. 4

    Björklund S, Skog S, Tribukait B, Thelander L. S-phase-specific expression of mammalian ribonucleotide reductase R1 and R2 subunit mRNAs. Biochemistry 29:5452–5458;1990.

  5. 5

    Björklund S, Hjortsberg K, Johansson E, Thelander L. Structure and promoter characterization of the gene encoding the large subunit (R1 protein) of mouse ribonucleotide reductase. Proc Natl Acad Sci USA 90:11322–11326;1993.

  6. 6

    Chang CH, Cheng YC. Substrate specificity of human ribonucleotide reductase from Molt-4F cells. Cancer Res 39:5081–5086;1979.

  7. 7

    Choy BK, McClarty GA, Wright JA. Transient elevation of ribonucleotide reductase activity, M2 mRNA and M2 protein in BALA/c 3T3 fibroblasts in the presence of 12-0-tetradecanoylphorbol-13-acetate. Biochem Biophys Res Commun 162:1417–1424;1989.

  8. 8

    Cory JG, Sato A. Regulation of ribonucleotide reductase activity in mammalian cells. Mol Cell Biochem 53:257–266;1983.

  9. 9

    Desai P, Ramakrishnan R, Lin ZW, Osak B, Glorioso JC, Levine M. The RR1 gene of herpes simplex virus type 1 is uniquely transactivated by ICP0 during infection. J Virology 67:6125–6135;1993.

  10. 10

    Engstrom Y, Eriksson S, Jildevik I, Skog S, Thelander L, Tribukait B. Cell cycle-dependent expression of mammalian ribonucleotide reductase: Differential regulation of the two subunits. J Biol Chem 260:9114–9116;1985.

  11. 11

    Goulian M, Beck WS. Purification and properties of cobamide-dependent ribonucleotide reductase fromLactobacillus leichmannii. J Biol Chem 241:4233–4242;1966.

  12. 12

    Howell ML, Sanders-Loehr J, Loehr TM, Roseman NA, Mathews CK, Slabaugh MB. Cloning of the Vaccinia virus ribonucleotide reductase small subunit gene. J Biol Chem 267:1705–1711;1992.

  13. 13

    Hurta RAR, Wright JA. Mammalian drug resistant mutants with multiple gene amplification: Genes encoding the M1 component of ribonucleotide reductase, the M2 component of ribonucleotide reductase, ornithine decarboxylase, p5–8, the H-subunit of ferritin and the L-subunit of ferritin. Biochem Biophys Acta 1087:165–172;1990.

  14. 14

    Hurta RA, Samuel SK, Greenberg AH, Wright JA. Early detection of ribonucleotide reductase gene expression by transforming growth factor beta-1 in malignant H-ras transformed cell lines. J Biol Chem 266:24097–24100;1991.

  15. 15

    Hurta RA, Wright JA. Alterations in the activity and regulation of mammalian ribonucleotide reductase by chlorambucil, a DNA damaging agent. J Biol Chem 267:7066–7071;1992.

  16. 16

    Jones C, Zhu F, Dhanwada KR. Analysis of a herpes simplex virus 2 fragment from the open reading frame of the large subunit of ribonucleotide reductase with transcriptional regulatory activity. DNA and Cell Biol 12:127–137;1993.

  17. 17

    Lankinen H, Everett R, Cross A, Conner J, Marsden HS. Epitope mapping identifies an exposed loop between the unique amino- and conserved carboxy-domains of the large subunit of herpes simplex virus type 1 ribonucleotide reductase. J Gen Virology 74:1871–1877;1993.

  18. 18

    Luo JA, Smith CC, Kulka M, Aurelian L. A truncated protein kinase domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) expressed inEscherichia coli. J Biol Chem 266:20976–20983;1991.

  19. 19

    Mann GJ, Musgrove EA, Fox RM, Thelander L. Ribonucleotide reductase M1 subunit in cellular proliferation, quiescence, and differentiation. Cancer Res 48:5151–5156;1988.

  20. 20

    McClarty GA, Chan AKM, Wright JA. Hydroxyurea-induced conversion of mammalian ribonucleotide reductase to a form hypersensitive to bleomycin. Cancer Res 46:4516–4521;1986.

  21. 21

    Moore EC, Reichard P. Enzymatic synthesis of deoxyribonucleotides. VI. The cytidine diphosphate reductase system from Novikoff hepatoma. J Biol Chem 239:3453–3456;1964.

  22. 22

    Reichard P, Baldesten A, Rutberg L. Formation of deoxycytidine phosphates from cytidine phosphates in extracts forEscherichia coli. J Biol Chem 236:1150–1157;1961.

  23. 23

    Reynolds CP, Biedler JL, Spengler BA, Reynolds DA, Ross RA, Frenkel EP, Smith RG. Characterization of human neuroblastoma cell lines established before and after therapy. J National Cancer Institute 76:375–387;1986.

  24. 24

    Salem JS, Scott CP, Li LH, Cooperman BS, Rubin H. High level expression of the large subunit of mouse ribonucleotide reductase in a baculovirus system. FEBS 323:93–95;1993.

  25. 25

    Srinivasan PR, Tonin PN, Wensing EJ, Lewis WH. The gene for ornithine decarboxylase is co-amplified in hydroxyurea-resistant hamster cells. J Biol Chem 262:12871–12878;1987.

  26. 26

    Steeper JR, Steuart CD. A rapid assay for CDP reductase activity in mammalian cell extracts. Anal Biochem 34:123–130;1970.

  27. 27

    Tagger AY, Wright JA. Molecular and cellular characterization of drug resistant hamster cell lines with alterations in ribonucleotide reductase. Int J Cancer 42:760–766;1988.

  28. 28

    Thelander M, Graslund A, Thelander L. Subunit M2 of mammalian ribonucleotide reductase. J Biol Chem 260:2737–2741;1985.

  29. 29

    Thelander L, Berg P. Isolation and characterization of expressible cDNA clones encoding the M1 and M2 subunits of mouse ribonucleotide reductase. Mol Cell Biol 6:3433–3442;1986.

  30. 30

    Wright JA, Hards RG, Dick JE. Studies of ribonucleotide reductase activity in intact permeabilized cells: A genetic approach. Adv Enzyme Regul 19:105–127;1981.

  31. 31

    Yen Y, Grill SP, Dutschman GE, Chang CN, Zhou BS, Cheng YC. Characterization of a hydroxyurea-resistant human KB cell line with supersensitivity to 6-thioguanine. Cancer Res 54:3686–3691;1994.

  32. 32

    Zhou BS, Hsu NY, Pan BC, Doroshow JH, Yen Y. Overexpression of ribonucleotide reductase in transfected human KB cells increases their resistance to hydroxyurea: M2 but not M1 is sufficient to increase resistance to hydroxyurea in transfected cells. Cancer Res 55:1328–1333;1995.

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Jong, A.Y., Yu, K., Zhou, B. et al. A simple and sensitive ribonucleotide reductase assay. J Biomed Sci 5, 62–68 (1998). https://doi.org/10.1007/BF02253357

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Key Words

  • Ribonucleotide reductase
  • Enzyme assay