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. 1993 Nov;12(11):4425–4431. doi: 10.1002/j.1460-2075.1993.tb06127.x

dUTP pyrophosphatase is an essential enzyme in Saccharomyces cerevisiae.

M H Gadsden 1, E M McIntosh 1, J C Game 1, P J Wilson 1, R H Haynes 1
PMCID: PMC413740  PMID: 8223452

Abstract

dUTP pyrophosphatase (dUTPase; EC 3.6.1.23) catalyses the hydrolysis of dUTP to dUMP and PPi and thereby prevents the incorporation of uracil into DNA during replication. Although it is widely believed that dUTPase is essential for cell viability because of this role, direct evidence supporting this assumption has not been presented for any eukaryotic system. We have analysed the role of dUTPase (DUT1) in the life cycle of yeast. Using gene disruption and tetrad analysis, we find that DUT1 is necessary for the viability of S. cerevisiae; however, under certain conditions dut1 null mutants survive if supplied with exogenous thymidylate (dTMP). Analyses with isogenic uracil-DNA-glycosylase (UNG1) deficient or proficient strains indicate that in the absence of dUTPase, cell death results from the incorporation of uracil into DNA and the attempted repair of this damage by UNG1-mediated excision repair. However, in dut1 ung1 double mutants, starvation for dTMP causes dividing cells to arrest and die in all phases of the cell cycle. This latter effect suggests that the extensive stable substitution of uracil for thymine in DNA leads to a general failure in macromolecular synthesis. These results are in general agreement with previous models in thymine-less death that implicate dUTP metabolism. They also suggest an alternative approach for chemotherapeutic drug design.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barclay B. J., Kunz B. A., Little J. G., Haynes R. H. Genetic and biochemical consequences of thymidylate stress. Can J Biochem. 1982 Mar;60(3):172–184. doi: 10.1139/o82-023. [DOI] [PubMed] [Google Scholar]
  2. Barclay B. J., Little J. G. Selection of yeast auxotrophs by thymidylate starvation. J Bacteriol. 1977 Dec;132(3):1036–1037. doi: 10.1128/jb.132.3.1036-1037.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chen D. C., Yang B. C., Kuo T. T. One-step transformation of yeast in stationary phase. Curr Genet. 1992 Jan;21(1):83–84. doi: 10.1007/BF00318659. [DOI] [PubMed] [Google Scholar]
  4. Dudley B., Hammond A., Deutsch W. A. The presence of uracil-DNA glycosylase in insects is dependent upon developmental complexity. J Biol Chem. 1992 Jun 15;267(17):11964–11967. [PubMed] [Google Scholar]
  5. Elder J. H., Lerner D. L., Hasselkus-Light C. S., Fontenot D. J., Hunter E., Luciw P. A., Montelaro R. C., Phillips T. R. Distinct subsets of retroviruses encode dUTPase. J Virol. 1992 Mar;66(3):1791–1794. doi: 10.1128/jvi.66.3.1791-1794.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  7. Goulian M., Bleile B., Tseng B. Y. Methotrexate-induced misincorporation of uracil into DNA. Proc Natl Acad Sci U S A. 1980 Apr;77(4):1956–1960. doi: 10.1073/pnas.77.4.1956. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Goulian M., Bleile B., Tseng B. Y. The effect of methotrexate on levels of dUTP in animal cells. J Biol Chem. 1980 Nov 25;255(22):10630–10637. [PubMed] [Google Scholar]
  9. 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]
  10. Ivarie R. Thymine methyls and DNA-protein interactions. Nucleic Acids Res. 1987 Dec 10;15(23):9975–9983. doi: 10.1093/nar/15.23.9975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Johnston L. H., Lowndes N. F. Cell cycle control of DNA synthesis in budding yeast. Nucleic Acids Res. 1992 May 25;20(10):2403–2410. doi: 10.1093/nar/20.10.2403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kohalmi S. E., Glattke M., McIntosh E. M., Kunz B. A. Mutational specificity of DNA precursor pool imbalances in yeast arising from deoxycytidylate deaminase deficiency or treatment with thymidylate. J Mol Biol. 1991 Aug 20;220(4):933–946. doi: 10.1016/0022-2836(91)90364-c. [DOI] [PubMed] [Google Scholar]
  13. Little J. G., Haynes R. H. Isolation and characterization of yeast mutants auxotrophic for 2'-deoxythymidine 5'-monophosphate. Mol Gen Genet. 1979 Jan 10;168(2):141–151. doi: 10.1007/BF00431440. [DOI] [PubMed] [Google Scholar]
  14. Lundberg L. G., Karlström O. H., Nyman P. O., Neuhard J. Isolation and characterization of the dut gene of Escherichia coli. I. Cloning in thermoinducible plasmids. Gene. 1983 Apr;22(1):115–126. doi: 10.1016/0378-1119(83)90070-7. [DOI] [PubMed] [Google Scholar]
  15. McGeoch D. J. Protein sequence comparisons show that the 'pseudoproteases' encoded by poxviruses and certain retroviruses belong to the deoxyuridine triphosphatase family. Nucleic Acids Res. 1990 Jul 25;18(14):4105–4110. doi: 10.1093/nar/18.14.4105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. McIntosh E. M., Ager D. D., Gadsden M. H., Haynes R. H. Human dUTP pyrophosphatase: cDNA sequence and potential biological importance of the enzyme. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):8020–8024. doi: 10.1073/pnas.89.17.8020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McIntosh E. M., Gadsden M. H., Haynes R. H. Transcription of genes encoding enzymes involved in DNA synthesis during the cell cycle of Saccharomyces cerevisiae. Mol Gen Genet. 1986 Sep;204(3):363–366. doi: 10.1007/BF00331011. [DOI] [PubMed] [Google Scholar]
  18. McIntosh E. M., Haynes R. H. Isolation of a Saccharomyces cerevisiae mutant strain deficient in deoxycytidylate deaminase activity and partial characterization of the enzyme. J Bacteriol. 1984 May;158(2):644–649. doi: 10.1128/jb.158.2.644-649.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. McIntosh E. M., Kunz B. A., Haynes R. H. Inhibition of DNA replication in Saccharomyces cerevisiae by araCMP. Curr Genet. 1986;10(8):579–585. doi: 10.1007/BF00418124. [DOI] [PubMed] [Google Scholar]
  20. McIntosh E. M., Ord R. W., Storms R. K. Transcriptional regulation of the cell cycle-dependent thymidylate synthase gene of Saccharomyces cerevisiae. Mol Cell Biol. 1988 Nov;8(11):4616–4624. doi: 10.1128/mcb.8.11.4616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mercer A. A., Fraser K. M., Stockwell P. A., Robinson A. J. A homologue of retroviral pseudoproteases in the parapoxvirus, orf virus. Virology. 1989 Oct;172(2):665–668. doi: 10.1016/0042-6822(89)90212-2. [DOI] [PubMed] [Google Scholar]
  22. Meyer-Siegler K., Mauro D. J., Seal G., Wurzer J., deRiel J. K., Sirover M. A. A human nuclear uracil DNA glycosylase is the 37-kDa subunit of glyceraldehyde-3-phosphate dehydrogenase. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8460–8464. doi: 10.1073/pnas.88.19.8460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mortimer R. K., Contopoulou C. R., King J. S. Genetic and physical maps of Saccharomyces cerevisiae, Edition 11. Yeast. 1992 Oct;8(10):817–902. doi: 10.1002/yea.320081002. [DOI] [PubMed] [Google Scholar]
  24. Muller S. J., Caradonna S. Cell cycle regulation of a human cyclin-like gene encoding uracil-DNA glycosylase. J Biol Chem. 1993 Jan 15;268(2):1310–1319. [PubMed] [Google Scholar]
  25. Nasmyth K. A., Tatchell K. The structure of transposable yeast mating type loci. Cell. 1980 Mar;19(3):753–764. doi: 10.1016/s0092-8674(80)80051-1. [DOI] [PubMed] [Google Scholar]
  26. Nation M. D., Guzder S. N., Giroir L. E., Deutsch W. A. Control of Drosophila deoxyuridine triphosphatase. Existence of a developmentally expressed protein inhibitor. Biochem J. 1989 Apr 15;259(2):593–596. doi: 10.1042/bj2590593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pri-Hadash A., Hareven D., Lifschitz E. A meristem-related gene from tomato encodes a dUTPase: analysis of expression in vegetative and floral meristems. Plant Cell. 1992 Feb;4(2):149–159. doi: 10.1105/tpc.4.2.149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pu W. T., Struhl K. Uracil interference, a rapid and general method for defining protein-DNA interactions involving the 5-methyl group of thymines: the GCN4-DNA complex. Nucleic Acids Res. 1992 Feb 25;20(4):771–775. doi: 10.1093/nar/20.4.771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pyles R. B., Sawtell N. M., Thompson R. L. Herpes simplex virus type 1 dUTPase mutants are attenuated for neurovirulence, neuroinvasiveness, and reactivation from latency. J Virol. 1992 Nov;66(11):6706–6713. doi: 10.1128/jvi.66.11.6706-6713.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Richards R. G., Sowers L. C., Laszlo J., Sedwick W. D. The occurrence and consequences of deoxyuridine in DNA. Adv Enzyme Regul. 1984;22:157–185. doi: 10.1016/0065-2571(84)90013-x. [DOI] [PubMed] [Google Scholar]
  31. Russel M., Kidd S., Kelley M. R. An improved filamentous helper phage for generating single-stranded plasmid DNA. Gene. 1986;45(3):333–338. doi: 10.1016/0378-1119(86)90032-6. [DOI] [PubMed] [Google Scholar]
  32. Slabaugh M. B., Roseman N. A. Retroviral protease-like gene in the vaccinia virus genome. Proc Natl Acad Sci U S A. 1989 Jun;86(11):4152–4155. doi: 10.1073/pnas.86.11.4152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Strahler J. R., Zhu X. X., Hora N., Wang Y. K., Andrews P. C., Roseman N. A., Neel J. V., Turka L., Hanash S. M. Maturation stage and proliferation-dependent expression of dUTPase in human T cells. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):4991–4995. doi: 10.1073/pnas.90.11.4991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Taylor A. F., Weiss B. Role of exonuclease III in the base excision repair of uracil-containing DNA. J Bacteriol. 1982 Jul;151(1):351–357. doi: 10.1128/jb.151.1.351-357.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Threadgill D. S., Steagall W. K., Flaherty M. T., Fuller F. J., Perry S. T., Rushlow K. E., Le Grice S. F., Payne S. L. Characterization of equine infectious anemia virus dUTPase: growth properties of a dUTPase-deficient mutant. J Virol. 1993 May;67(5):2592–2600. doi: 10.1128/jvi.67.5.2592-2600.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tsao S. G., Brunk C. F., Pearlman R. E. Hybridization of nucleic acids directly in agarose gels. Anal Biochem. 1983 Jun;131(2):365–372. doi: 10.1016/0003-2697(83)90185-9. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Wang L., Weiss B. dcd (dCTP deaminase) gene of Escherichia coli: mapping, cloning, sequencing, and identification as a locus of suppressors of lethal dut (dUTPase) mutations. J Bacteriol. 1992 Sep;174(17):5647–5653. doi: 10.1128/jb.174.17.5647-5653.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Warner H. R., Duncan B. K., Garrett C., Neuhard J. Synthesis and metabolism of uracil-containing deoxyribonucleic acid in Escherichia coli. J Bacteriol. 1981 Feb;145(2):687–695. doi: 10.1128/jb.145.2.687-695.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. el-Hajj H. H., Wang L., Weiss B. Multiple mutant of Escherichia coli synthesizing virtually thymineless DNA during limited growth. J Bacteriol. 1992 Jul;174(13):4450–4456. doi: 10.1128/jb.174.13.4450-4456.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. el-Hajj H. H., Zhang H., Weiss B. Lethality of a dut (deoxyuridine triphosphatase) mutation in Escherichia coli. J Bacteriol. 1988 Mar;170(3):1069–1075. doi: 10.1128/jb.170.3.1069-1075.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

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