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. 1992 Jul;174(13):4450–4456. doi: 10.1128/jb.174.13.4450-4456.1992

Multiple mutant of Escherichia coli synthesizing virtually thymineless DNA during limited growth.

H H el-Hajj 1, L Wang 1, B Weiss 1
PMCID: PMC206231  PMID: 1624437

Abstract

The dut gene of Escherichia coli encodes deoxyuridine triphosphatase, an enzyme that prevents the incorporation of dUTP into DNA and that is needed in the de novo biosynthesis of thymidylate. We produced a conditionally lethal dut(Ts) mutation and isolated a phenotypic revertant that had a mutation in an unknown gene tentatively designated dus (for dut suppressor). The dus mutation restored the ability of the dut mutant to grow at 42 degrees C without restoring its enzymatic activity or thymidylate independence. A strain was constructed bearing, in addition to these mutations, ones affecting the following genes and their corresponding products: ung, which produces uracil-DNA N-glycosylase, a repair enzyme that removes uracil from DNA; deoA, which produces thymidine (deoxyuridine) phosphorylase, which would degrade exogenous deoxyuridine; and thyA, which produces thymidylate synthase. When grown at 42 degrees C in minimal medium containing deoxyuridine, the multiple mutant displayed a 93 to 96% substitution of uracil for thymine in new DNA. Growth stopped after the cellular DNA had increased 1.6- to 1.9-fold and the cell mass had increased 1.7- to 2.7-fold, suggesting a general failure of macromolecular biosynthesis. DNA hybridization confirmed that the uracil-containing DNA was chromosomal and that new rounds of initiation must have occurred during its synthesis.

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Selected References

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  1. Anderson R. P., Roth J. R. Tandem genetic duplications in phage and bacteria. Annu Rev Microbiol. 1977;31:473–505. doi: 10.1146/annurev.mi.31.100177.002353. [DOI] [PubMed] [Google Scholar]
  2. BURTON K. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J. 1956 Feb;62(2):315–323. doi: 10.1042/bj0620315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bachmann B. J. Linkage map of Escherichia coli K-12, edition 8. Microbiol Rev. 1990 Jun;54(2):130–197. doi: 10.1128/mr.54.2.130-197.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bochner B. R., Ames B. N. Complete analysis of cellular nucleotides by two-dimensional thin layer chromatography. J Biol Chem. 1982 Aug 25;257(16):9759–9769. [PubMed] [Google Scholar]
  5. Chan E., Weiss B. Endonuclease IV of Escherichia coli is induced by paraquat. Proc Natl Acad Sci U S A. 1987 May;84(10):3189–3193. doi: 10.1073/pnas.84.10.3189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clements J. E., Rogers S. G., Weiss B. A DNase for apurinic/apyrimidinic sites associated with exonuclease III of Hemophilus influenzae. J Biol Chem. 1978 May 10;253(9):2990–2999. [PubMed] [Google Scholar]
  7. Cohen S. S. On the nature of thymineless death. Ann N Y Acad Sci. 1971 Nov 30;186:292–301. doi: 10.1111/j.1749-6632.1971.tb31155.x. [DOI] [PubMed] [Google Scholar]
  8. Cunningham R. P., Saporito S. M., Spitzer S. G., Weiss B. Endonuclease IV (nfo) mutant of Escherichia coli. J Bacteriol. 1986 Dec;168(3):1120–1127. doi: 10.1128/jb.168.3.1120-1127.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Demple B., Linn S. On the recognition and cleavage mechanism of Escherichia coli endodeoxyribonuclease V, a possible DNA repair enzyme. J Biol Chem. 1982 Mar 25;257(6):2848–2855. [PubMed] [Google Scholar]
  10. Duncan B. K. Isolation of insertion, deletion, and nonsense mutations of the uracil-DNA glycosylase (ung) gene of Escherichia coli K-12. J Bacteriol. 1985 Nov;164(2):689–695. doi: 10.1128/jb.164.2.689-695.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. EIDLIC L., NEIDHARDT F. C. PROTEIN AND NUCLEIC ACID SYNTHESIS IN TWO MUTANTS OF ESCHERICHIA COLI WITH TEMPERATURE-SENSITIVE AMINOACYL RIBONUCLEIC ACID SYNTHETASES. J Bacteriol. 1965 Mar;89:706–711. doi: 10.1128/jb.89.3.706-711.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ferenci T., Kornberg H. L., Smith Janet. Isolation and properties of a regulatory mutant in the hexose phosphate transport system of Escherichia coli. FEBS Lett. 1971 Mar 5;13(3):133–136. doi: 10.1016/0014-5793(71)80218-1. [DOI] [PubMed] [Google Scholar]
  13. Grilley M., Welsh K. M., Su S. S., Modrich P. Isolation and characterization of the Escherichia coli mutL gene product. J Biol Chem. 1989 Jan 15;264(2):1000–1004. [PubMed] [Google Scholar]
  14. Hirota Y., Ryter A., Jacob F. Thermosensitive mutants of E. coli affected in the processes of DNA synthesis and cellular division. Cold Spring Harb Symp Quant Biol. 1968;33:677–693. doi: 10.1101/sqb.1968.033.01.077. [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. Hong J. S., Ames B. N. Localized mutagenesis of any specific small region of the bacterial chromosome. Proc Natl Acad Sci U S A. 1971 Dec;68(12):3158–3162. doi: 10.1073/pnas.68.12.3158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kohara Y., Akiyama K., Isono K. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell. 1987 Jul 31;50(3):495–508. doi: 10.1016/0092-8674(87)90503-4. [DOI] [PubMed] [Google Scholar]
  18. Kumagai M., Fujimoto M., Kuninaka A. Determination of base composition of DNA by high performance liquid chromatography of its nuclease P1 hydrolysate. Nucleic Acids Symp Ser. 1988;(19):65–68. [PubMed] [Google Scholar]
  19. Lindahl T. DNA repair enzymes. Annu Rev Biochem. 1982;51:61–87. doi: 10.1146/annurev.bi.51.070182.000425. [DOI] [PubMed] [Google Scholar]
  20. Lundberg L. G., Thoresson H. O., Karlström O. H., Nyman P. O. Nucleotide sequence of the structural gene for dUTPase of Escherichia coli K-12. EMBO J. 1983;2(6):967–971. doi: 10.1002/j.1460-2075.1983.tb01529.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. MAALOE O., HANAWALT P. C. Thymine deficiency and the normal DNA replication cycle. I. J Mol Biol. 1961 Apr;3:144–155. doi: 10.1016/s0022-2836(61)80041-7. [DOI] [PubMed] [Google Scholar]
  22. Medoff G. Nucleic acid and protein synthesis during thymineless death in lysogenic and nonlysogenic thymine auxotrophs. J Bacteriol. 1972 Jan;109(1):462–464. doi: 10.1128/jb.109.1.462-464.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  24. Spitzer E. D., Weiss B. dfp Gene of Escherichia coli K-12, a locus affecting DNA synthesis, codes for a flavoprotein. J Bacteriol. 1985 Dec;164(3):994–1003. doi: 10.1128/jb.164.3.994-1003.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sutcliffe J. G. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):77–90. doi: 10.1101/sqb.1979.043.01.013. [DOI] [PubMed] [Google Scholar]
  26. Taylor A. F., Siliciano P. G., Weiss B. Cloning of the dut (deoxyuridine triphosphatase) gene of Escherichia coli. Gene. 1980 May;9(3-4):321–336. doi: 10.1016/0378-1119(90)90330-t. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Tsaneva I. R., Weiss B. soxR, a locus governing a superoxide response regulon in Escherichia coli K-12. J Bacteriol. 1990 Aug;172(8):4197–4205. doi: 10.1128/jb.172.8.4197-4205.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. White B. J., Hochhauser S. J., Cintron N. M., Weiss B. Genetic mapping of xthA, the structural gene for exonuclease III in Escherichia coli K-12. J Bacteriol. 1976 Jun;126(3):1082–1088. doi: 10.1128/jb.126.3.1082-1088.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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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