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. 1980 Apr;77(4):1956–1960. doi: 10.1073/pnas.77.4.1956

Methotrexate-induced misincorporation of uracil into DNA

M Goulian 1, B Bleile 1, B Y Tseng 1
PMCID: PMC348628  PMID: 6929529

Abstract

A line of human lymphoid cells was tested for the presence of dUMP in DNA with or without treatment with the dihydrofolate reductase inhibitor, methotrexate. Cells treated with methotrexate and labeled with [3H]dUrd contained dUMP in DNA in readily detectable amounts (≈0.8 pmol of dUMP per μmol of total DNA nucleotide), and this was increased ≈3-fold if the cells were also treated with Ura at the same time. No dUMP (<1 fmol/μmol of DNA) could be detected by these methods in DNA from cells not treated with methotrexate, regardless of whether Ura was present or absent. The presence of dUMP in DNA from cells treated with methotrexate is a result of the great increase in intracellular concentration of dUTP and the fall in dTTP that accompany inhibition of thymidylate synthetase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase; EC 2.1.1.45) by the drug. These changes are apparently sufficient to overcome the normal mechanisms that exclude dUMP from DNA, and the enhancement by Ura reflects suppression of one of the mechanisms, Ura removal from DNA by the enzyme Ura-DNA glycosylase. The results suggest an active lesion of DNA in cells in which thymidylate synthetase is inhibited. Under these conditions there appears to be a cyclic incorporation and removal of dUMP resulting from reinsertion of dUMP during gap repair at sites of Ura removal. This consequence of the normal excision-repair process, which occurs when intracellular levels of dUTP approach those of dTTP, may have effects related to the cytotoxicity of drug inhibitors of thymidylate synthetase, clinical deficiencies of folate and vitamin B-12, and thymineless death, in general.

Keywords: thymidylate synthetase, intracellular dUTP, dTTP, DNA repair, cytotoxicity, thymineless death

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1959

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Selected References

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  1. BERTANI L. E., HAEGGMARK A., REICHARD P. ENZYMATIC SYNTHESIS OF DEOXYRIBONUCLEOTIDES. II. FORMATION AND INTERCONVERSION OF DEOXYURIDINE PHOSPHATES. J Biol Chem. 1963 Oct;238:3407–3413. [PubMed] [Google Scholar]
  2. Bjursell G., Reichard P. Effects of thymidine on deoxyribonucleoside triphosphate pools and deoxyribonucleic acid synthesis in Chinese hamster ovary cells. J Biol Chem. 1973 Jun 10;248(11):3904–3909. [PubMed] [Google Scholar]
  3. Brynolf K., Eliasson R., Reichard P. Formation of Okazaki fragments in polyoma DNA synthesis caused by misincorporation of uracil. Cell. 1978 Mar;13(3):573–580. doi: 10.1016/0092-8674(78)90330-6. [DOI] [PubMed] [Google Scholar]
  4. Cohen S. S., Barner H. D. STUDIES ON UNBALANCED GROWTH IN ESCHERICHIA COLI. Proc Natl Acad Sci U S A. 1954 Oct;40(10):885–893. doi: 10.1073/pnas.40.10.885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cone R., Duncan J., Hamilton L., Friedberg E. C. Partial purification and characterization of a uracil DNA N-glycosidase from Bacillus subtilis. Biochemistry. 1977 Jul 12;16(14):3194–3201. doi: 10.1021/bi00633a024. [DOI] [PubMed] [Google Scholar]
  6. Dube D. K., Kunkel T. A., Seal G., Loeb L. A. Distinctive properties of mammalian DNA polymerases. Biochim Biophys Acta. 1979 Feb 27;561(2):369–382. doi: 10.1016/0005-2787(79)90145-x. [DOI] [PubMed] [Google Scholar]
  7. Ensminger W. D., Frei E., 3rd The prevention of methotrexate toxicity by thymidine infusions in humans. Cancer Res. 1977 Jun;37(6):1857–1863. [PubMed] [Google Scholar]
  8. Freifelder D., Katz G. Persistence of small fragments of newly synthesized DNA in bacteria following thymidine starvation. J Mol Biol. 1971 Apr 28;57(2):351–354. doi: 10.1016/0022-2836(71)90351-2. [DOI] [PubMed] [Google Scholar]
  9. Freifelder D. Single-strand breaks in bacterial DNA associated with thymine starvation. J Mol Biol. 1969 Oct 14;45(1):1–7. doi: 10.1016/0022-2836(69)90205-8. [DOI] [PubMed] [Google Scholar]
  10. Fridland A. Effect of methotrexate on deoxyribonucleotide pools and DNA synthesis in human lymphocytic cells. Cancer Res. 1974 Aug;34(8):1883–1888. [PubMed] [Google Scholar]
  11. GREENBERG G. R., SOMERVILLE R. L. Deoxyuridylate kinase activity and deoxyuridinetriphosphatase in Escherichia coli. Proc Natl Acad Sci U S A. 1962 Feb;48:247–257. doi: 10.1073/pnas.48.2.247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gallo R. C., Perry S. The enzymatic mechanisms for deoxythymidine synthesis in human leukocytes. IV. Comparisons between normal and leukemic leukocytes. J Clin Invest. 1969 Jan;48(1):105–116. doi: 10.1172/JCI105958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Grafstrom R. H., Tseng B. Y., Goulian M. The incorporation of uracil into animal cell DNA in vitro. Cell. 1978 Sep;15(1):131–140. doi: 10.1016/0092-8674(78)90089-2. [DOI] [PubMed] [Google Scholar]
  14. Grindey G. R., Nichol C. A. Mammalian deoxyuridine 5'-triphosphate pyrophosphatase. Biochim Biophys Acta. 1971 Jun 30;240(2):180–183. doi: 10.1016/0005-2787(71)90655-1. [DOI] [PubMed] [Google Scholar]
  15. Hochhauser S. J., Weiss B. Escherichia coli mutants deficient in deoxyuridine triphosphatase. J Bacteriol. 1978 Apr;134(1):157–166. doi: 10.1128/jb.134.1.157-166.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Isacoff W. H., Morrison P. F., Aroesty J., Willis K. L., Block J. B., Lincoln T. L. Pharmacokinetics of high-dose methotrexate with citrovorum factor rescue. Cancer Treat Rep. 1977 Dec;61(9):1665–1674. [PubMed] [Google Scholar]
  17. Jackson R. C. The regulation of thymidylate biosynthesis in Novikoff hepatoma cells and the effects of amethopterin, 5-fluorodeoxyuridine, and 3-deazauridine. J Biol Chem. 1978 Oct 25;253(20):7440–7446. [PubMed] [Google Scholar]
  18. KRENITSKY T. A., MELLORS J. W., BARCLAY R. K. PYRIMIDINE NUCLEOSIDASES. THEIR CLASSIFICATION AND RELATIONSHIP TO URIC ACID RIBONUCLEOSIDE PHOSPHORYLASE. J Biol Chem. 1965 Mar;240:1281–1286. [PubMed] [Google Scholar]
  19. Krstulovic A. M., Brown P. R., Rosie D. M. Identification of nucleosides and bases in serum and plasma samples by reverse-phase high performance liquid chromatography. Anal Chem. 1977 Dec;49(14):2237–2241. doi: 10.1021/ac50022a032. [DOI] [PubMed] [Google Scholar]
  20. Lindahl T. DNA glycosylases, endonucleases for apurinic/apyrimidinic sites, and base excision-repair. Prog Nucleic Acid Res Mol Biol. 1979;22:135–192. doi: 10.1016/s0079-6603(08)60800-4. [DOI] [PubMed] [Google Scholar]
  21. Lindahl T., Ljungquist S., Siegert W., Nyberg B., Sperens B. DNA N-glycosidases: properties of uracil-DNA glycosidase from Escherichia coli. J Biol Chem. 1977 May 25;252(10):3286–3294. [PubMed] [Google Scholar]
  22. MALEY F., MALEY G. F. On the nature of a sparing effect by thymidine on the utilization of deoxycytidine. Biochemistry. 1962 Sep;1:847–851. doi: 10.1021/bi00911a017. [DOI] [PubMed] [Google Scholar]
  23. MENNIGMANN H. D., SZYBALSKI W. Molecular mechanism of thymine-less death. Biochem Biophys Res Commun. 1962 Nov 27;9:398–404. doi: 10.1016/0006-291x(62)90023-2. [DOI] [PubMed] [Google Scholar]
  24. Makino F., Munakata N. Deoxyuridine residues in DNA of thymine-requiring Bacillus subtilis strains with defective N-glycosidase activity for uracil-containing DNA. J Bacteriol. 1978 Apr;134(1):24–29. doi: 10.1128/jb.134.1.24-29.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Moore E. C., Hurlbert R. B. Regulation of mammalian deoxyribonucleotide biosynthesis by nucleotides as activators and inhibitors. J Biol Chem. 1966 Oct 25;241(20):4802–4809. [PubMed] [Google Scholar]
  26. Myers C. E., Young R. C., Chabner B. A. Biochemical determinants of 5-fluorouracil response in vivo. The role of deoxyuridylate pool expansion. J Clin Invest. 1975 Nov;56(5):1231–1238. doi: 10.1172/JCI108199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nakayama H., Hanawalt P. Sedimentation analysis of deoxyribonucleic acid from thymine-starved Escherichia coli. J Bacteriol. 1975 Feb;121(2):537–547. doi: 10.1128/jb.121.2.537-547.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Neuhard J., Thomassen E. Altered deoxyribonucleotide pools in P2 eductants of Escherichia coli K-12 due to deletion of the dcd gene. J Bacteriol. 1976 May;126(2):999–1001. doi: 10.1128/jb.126.2.999-1001.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. O'Donovan G. A., Edlin G., Fuchs J. A., Neuhard J., Thomassen E. Deoxycytidine triphosphate deaminase: characterization of an Escherichia coli mutant deficient in the enzyme. J Bacteriol. 1971 Feb;105(2):666–672. doi: 10.1128/jb.105.2.666-672.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Olivera B. M., Manlapaz-Ramos P., Warner H. R., Duncan B. K. DNA intermediates at the Escherichia coli replication fork. II. Studies using dut and ung mutants in vitro. J Mol Biol. 1979 Mar 5;128(3):265–275. doi: 10.1016/0022-2836(79)90087-1. [DOI] [PubMed] [Google Scholar]
  31. Perlman D., Huberman J. A. Asymmetric Okazaki piece synthesis during replication of simian virus 40 DNA in vivo. Cell. 1977 Dec;12(4):1029–1043. doi: 10.1016/0092-8674(77)90167-2. [DOI] [PubMed] [Google Scholar]
  32. Salzman N. P., Thoren M. M. Inhibition in the joining of DNA intermediates to growing simian virus 40 chains. J Virol. 1973 May;11(5):721–729. doi: 10.1128/jvi.11.5.721-729.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sekiguchi M., Hayakawa H., Makino F., Tanaka K., Okada Y. A human enzyme that liberates uracil from DNA. Biochem Biophys Res Commun. 1976 Nov 22;73(2):293–299. doi: 10.1016/0006-291x(76)90706-3. [DOI] [PubMed] [Google Scholar]
  34. Shlomai J., Kornberg A. Deoxyuridine triphosphatase of Escherichia coli. Purification, properties, and use as a reagent to reduce uracil incorporation into DNA. J Biol Chem. 1978 May 10;253(9):3305–3312. [PubMed] [Google Scholar]
  35. Skoog L., Nordenskjöld B., Humla S., Hägerström T. Effects of methotrexate on deoxyribonucleotide pools and nucleic acid synthesis in human osteosarcoma cells. Eur J Cancer. 1976 Oct;12(10):839–845. doi: 10.1016/0014-2964(76)90100-6. [DOI] [PubMed] [Google Scholar]
  36. Talpaert-Borlé M., Clerici L., Campagnari F. Isolation and characterization of a uracil-DNA glycosylase from calf thymus. J Biol Chem. 1979 Jul 25;254(14):6387–6391. [PubMed] [Google Scholar]
  37. Tamanoi F., Okazaki T. Uracil incorporation into nascent DNA of thymine-requiring mutant of Bacillus subtilis 168. Proc Natl Acad Sci U S A. 1978 May;75(5):2195–2199. doi: 10.1073/pnas.75.5.2195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tattersall M. H., Harrap K. R. Changes in the deoxyribonucleoside triphosphate pools of mouse 5178Y lymphoma cells following exposure to methotrexate or 5-fluorouracil. Cancer Res. 1973 Dec;33(12):3086–3090. [PubMed] [Google Scholar]
  39. Tattersall M. H., Jackson R. C., Connors T. A., Harrap K. R. Combination chemotherapy: the interaction of methotrexate and 5-fluorouracil. Eur J Cancer. 1973 Oct;9(10):733–739. doi: 10.1016/0014-2964(73)90064-9. [DOI] [PubMed] [Google Scholar]
  40. Tseng B. Y., Goulian M. DNA synthesis in human lymphocyts: intermediates in DNA synthesis, in vitro and in vivo. J Mol Biol. 1975 Dec 5;99(2):317–337. doi: 10.1016/s0022-2836(75)80149-5. [DOI] [PubMed] [Google Scholar]
  41. Tseng B. Y., Grafstrom R. H., Revie D., Oertel W., Goulian M. Studies on early intermediates in the synthesis of DNA in animal cells. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):263–270. doi: 10.1101/sqb.1979.043.01.032. [DOI] [PubMed] [Google Scholar]
  42. Tye B. K., Chien J., Lehman I. R., Duncan B. K., Warner H. R. Uracil incorporation: a source of pulse-labeled DNA fragments in the replication of the Escherichia coli chromosome. Proc Natl Acad Sci U S A. 1978 Jan;75(1):233–237. doi: 10.1073/pnas.75.1.233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tye B. K., Lehman I. R. Excision repair of uracil incorporated in DNA as a result of a defect in dUTPase. J Mol Biol. 1977 Dec 5;117(2):293–306. doi: 10.1016/0022-2836(77)90128-0. [DOI] [PubMed] [Google Scholar]
  44. Tye B. K., Nyman P. O., Lehman I. R., Hochhauser S., Weiss B. Transient accumulation of Okazaki fragments as a result of uracil incorporation into nascent DNA. Proc Natl Acad Sci U S A. 1977 Jan;74(1):154–157. doi: 10.1073/pnas.74.1.154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Walker J. R. Thymine Starvation and Single-Strand Breaks in Chromosomal Deoxyribonucleic acid of Escherichia coli. J Bacteriol. 1970 Dec;104(3):1391–1392. doi: 10.1128/jb.104.3.1391-1392.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Williams M. V., Cheng Y. Human deoxyuridine triphosphate nucleotidohydrolase. Purification and characterization of the deoxyuridine triphosphate nucleotidohydrolase from acute lymphocytic leukemia. J Biol Chem. 1979 Apr 25;254(8):2897–2901. [PubMed] [Google Scholar]
  47. Wist E., Unhjem O., Krokan H. Accumulation of small fragments of DNA in isolated HeLa cell nuclei due to transient incorporation of dUMP. Biochim Biophys Acta. 1978 Sep 27;520(2):253–270. doi: 10.1016/0005-2787(78)90225-3. [DOI] [PubMed] [Google Scholar]
  48. Yoshinaga K. Double-strand scission of DNA involved in thymineless death of Escherichia coli 15 TAU. Biochim Biophys Acta. 1973 Jan 19;294(1):204–213. [PubMed] [Google Scholar]

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