Abstract
The polymerization of nucleotide analogs into DNA is a common strategy used to inhibit DNA synthesis in rapidly dividing tumor cells and viruses. The mammalian DNA polymerases catalyze the insertion of the arabinofuranosyl analogs of dNTPs (aranucleotides) into DNA efficiently, but elongate from the 3′ aranucleotides poorly. Slow elongation provides an opportunity for exonucleases to remove aranucleotides. The exonuclease activity associated with DNA polymerase δ removes araCMP from 3′ termini with the same efficiency that it removes a paired 3′ deoxycytosine suggesting that the proofreading exonucleases associated with DNA polymerases might remove aranucleotides inefficiently. A separate 30 kDa exonuclease has been purified from mammalian cells that removes araCMP from 3′ termini. The activity of this enzyme in the cell could remove aranucleotides from 3′ termini of DNA and decrease the efficacy of the analogs. Inhibition analysis of the purified exonuclease shows that this enzyme is inhibited by thioinosine monophosphate (TIMP) with aK i=17 μM. When high TIMP levels are generated in HL-60 cells, incorporation of araC in DNA is increased about 16-fold relative to total DNA synthesis. This increased araC in DNA is likely a result of exonuclease inhibition in the cell. Thus, exonucleases in cells might play an important role in removing aranucleotides inserted by DNA polymerases.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Goodman, M. F., Creighton, S., Bloom, L. B., and Petruska, J. (1993) Biochemical basis of DNA replication fidelity.Crit. Rev. Biochem. Mol. Biol. 28, 83–126.
Schaaper, R. M. (1993) Base selection, proofreading, and mismatch repair during DNA replication inEscherichia coli.J. Biol. Chem. 268, 23,762–23,765.
Goodman, M. F. (1997) Hydrogen bonding revisited: geometric selection as a principal determinant of DNA replication fidelity.Proc. Natl. Acad. Sci. USA 94, 10,493–10,495.
Gao, Y. G., Van der Marel, G. A., Van Boom, J. H., and Wang, A. H. J. (1991) Molecular structure of a DNA decamer containing an anticancer nucleoside arabinosylcytosine: conformational perturbation by arabinosylcytosine in B-DNA.Biochemistry 30, 9922–9931.
Schweitzer, B. I., Mikita, T., Kellogg, G. W., Gardner, K. H., and Beardsley, G. P. (1994) Solution structure of a DNA dodecamer containing the anti-neoplastic agent arabinosylcytosine: combined use of NMR, restrained molecular dynamics, and full relaxation matrix refinement.Biochemistry.33, 11,460–11,475.
Townsend, A. and Cheng, Y. C. (1987) Sequence-specific effects of ara-5-aza-CTP and ara-CTP on DNA synthesis by purified human DNA polymerasesin vitro: visualization of chain elongation on a defined template.Mol. Pharmacol. 32, 330–339.
Ohno, Y., Spriggs, D., Matsukage, A., Ohno, T. and Kufe, D. (1988) Effects of 1-β-D-arabinofuranosycytosine incorporation on elongation of specific DNA sequences by DNA polymerase-β.Cancer Res. 48, 1494–1498.
Reid, R., Mar, E. C., Huang, E. S., and Topal, M. D. (1988) Insertion and extension of acyclic, dideoxy, and aranucleotides by herpesviridae, human a and human β-polymerases. A unique inhibition mechanism for 9-(1,3-dihydroxy-2-propoxymethyl)guanine triphosphate.J. Biol. Chem. 263, 3898–3904.
Perrino, F. W. and Mekosh, H. L. (1992) Incorporation of cytosine arabinoside monophosphate into DNA at internucleotide linkages by human DNA polymerase-a.J. Biol. Chem. 267, 23,043–23,051.
Kornberg, A. and Baker, T. (1992)DNA Replication. 2nd ed., Freeman, New York.
Lee, M. Y. W. T., Byrnes, J. J., Downey, K. M., and So, A. G. (1980) Mechanism of inhibition of deoxyribonucleic acid synthesis by 1-β-D-arabinofuranosyladenosine triphosphate and its potentiation by 6-mercaptopurine ribonucleoside 5′-monophosphate.Biochemistry 19, 215–219.
Wright, G. E. and Brown, N. C. (1990) Deoxyribonucleotide analogs as inhibitors and substrates of DNA polymerases.Pharmac. Ther. 47, 447–497.
Major, P. P., Egan, E. M., Beardsley, G. P., Minden, M. D., and Kufe, D. W. (1981) Lethality of human myeloblasts correlates with the incorporation of arabinofuranosylcytosine into DNA.Proc. Natl. Acad. Sci. USA 78, 3235–3239.
Kufe, D. W., Major, P. P., Egan, E. M. and Beardsley, G. P. (1980) Correlation of cytotoxicity with incorporation of ara-C into DNA.J. Biol. Chem. 255, 8997–9000.
Syväoja, J., Suomensaari, S., Nishida, C., Goldsmith, J. S., Chui, G. S. J., Jain, S., and Linn, S. (1990) DNA polymerases alpha, delta, and epsilon: three distinct enzymes from HeLa cells.Proc. Nat. Acad. Sci. (USA) 87, 6664–6668.
Lewis, W., Meyer, R. R., Simpson, J. F., Colacino, J. M., and Perrino, F. W. (1994) Mammalian DNA polymerases α,β,γ,δ, and ε incorporate fialuridine (FIAU) monophosphate into DNA and are inhibited competitively by FIAU Triphosphate.Biochemistry 33, 14620–14624.
Perrino, F. W., Miller, H., and Ealey, K. A. (1994) Identification of a 3′→5′ exonuclease that removes cytosine arabinoside monophosphate from 3′ termini of DNA.J. Biol. Chem. 2669, 16,357–16,363.
Miller, H. and Perrino, F. W. (1996) Kinetic mechanism of the 3′→5′ proofreading exonuclease of DNA polymerase III. Analysis by steady state and pre-steady state methods.Biochemistry 35, 12,919–12,925.
Perrino, F. W. and Loeb, L. A., (1989) Differential extension of 3′ mispairs is a major contribution to the high fidelity of calf thymus DNA polymerase-alpha.J. Biol. Chem. 264, 2898–2905.
Blank, A., Sugiyama, R. H., and Dekker, C. A. (1982) Activity staining of nucleolytic enzymes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis: use of aqueous isopropanol to remove detergent from gels.Anal. Biochem. 120, 267–275.
Spanos, A. and Hubscher, U. (1983) Recovery of functional proteins in sodium dodecyl sulfate gels.Methods in Enzymology.91, 263–727.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature.227, 680–685.
Longley, M. J. and Mosbaugh, D. W. (1991) Characterization of DNA metabolizing enzymesin situ following polyacrylamide gel electrophoresis.Biochemistry 30, 2655–2664.
Zimm, S., Johnson, G. E., Chabner, B. A., and Poplack, D. G. (1985) Cellular pharmacokinetics of mercaptopurine in human neoplastic cells and cell lines.Cancer Res. 45, 4156–4161.
Ramilo-Torno, L. V., and Avramis, V. I. (1995) Intracellular pharmacodynamic studies of the synergistic combination of 6-mercaptopurine and cytosine arabinoside in human leukemia cells.Cancer Chemother. Pharmacol. 35, 191–199.
Ross, D. D., Chen, S. R. S., and Cuddy, D. P. (1990) Effects of 1-β-D-arabinofuranosylcytosine on DNA replication intermediates monitored by pH-step alkaline elution.Cancer Res. 50, 2658–2666.
Wills, P., Hickey, R., Ross, D., Cuddy, D., and Malkas, L. (1996) A novelin vitro model system for studying the action of ara-C.Cancer Chemother. Pharmacol. 38, 366–372.
Lindahl, T., Gally, J. A., and Edelman, G. M. (1969) Properties of deoxyribonuclease III from mammalian tissues.J. Biol. Chem. 244, 5014–5019.
Hollis, G. F. and Grossman, L. (1981) Purification and characterization of DNase VII, a 3′ leads to 5′-directed exonuclease from human placenta.J. Biol. Chem. 256, 8074–8079.
Skarnes, W., Bonin, P., and Baril, E. (1986) Exonuclease activity associated with a multiprotein form of HeLa cell DNA polymerase-α: purification and properties of the exonuclease. J. Biol. Chem.261, 6629–6636.
Skalski V., Liu, S. H., and Cheng, Y. C. (1995) Removal of anti-human immunodeficiency virus 2′,3′-dideoxynucleoside monophosphates from DNA by a novel human cytosolic 3′→5′ exonuclease.Biochem. Pharmacol. 50, 815–821.
Kamiya, K., Huang, P., and Plunkett, W. (1996) Inhibition of the 3′→5′ exonuclease of human DNA polymerase-ε by fludarabine-terminated DNA.J. Biol. Chem. 271, 19,428–19,435.
Gandhi, V., Legha, J., Chen, F., Hertel, L. W. and Plunkett, W. (1996) Excision of 2′, 2′-difluordeoxycytidine (gemcitabine) monophosphate residues from DNA.Cancer Res. 56, 4453–4459.
Major, P., Egan, E. M., Herrick, D., and Kufe, D. (1982) Effect of araC incorporation on deoxyribonucleic acid synthesis in cells.Biochem. Pharmacol. 31, 2937–2940.
Kufe, D. W., Munroe, D., Herrick, D., Egan, E. and Spriggs, D. (1984) Effects of 1-β-D-arabinofuranosylcytosine incorporation on eukaryotic DNA template function.Mol. Pharmacol. 26, 128–134.
Kufe, D., Spriggs, D., Egan, E. M. and Munroe, D. (1984) Relationships among ara-CTP pools, formation of (ara-C) DNA, and cytotoxicity of human leukemic cells.Blood 64, 54–58.
Zahn, R. K., Muller, W. E. G., Forster, W., Maidhof, A., and Beyer, R. (1972) Action of 1-β-D-arabinofuranosylcytosine on mammalian tumor cells-1. Incorporation into DNA.Europ. J. Cancer 8, 391–396.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Perrino, F.W., Mazur, D.J., Ward, H. et al. Exonucleases and the incorporation of aranucleotides into DNA. Cell Biochem Biophys 30, 331–352 (1999). https://doi.org/10.1007/BF02738118
Issue Date:
DOI: https://doi.org/10.1007/BF02738118