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. 1991 Aug 25;19(16):4473–4478. doi: 10.1093/nar/19.16.4473

Human uracil-DNA glycosylase complements E. coli ung mutants.

L C Olsen 1, R Aasland 1, H E Krokan 1, D E Helland 1
PMCID: PMC328636  PMID: 1886771

Abstract

We have previously isolated a cDNA encoding a human uracil-DNA glycosylase which is closely related to the bacterial and yeast enzymes. In vitro expression of this cDNA produced a protein with an apparent molecular weight of 34 K in agreement with the size predicted from the sequence data. The in vitro expressed protein exhibited uracil-DNA glycosylase activity. The close resemblance between the human and the bacterial enzyme raised the possibility that the human enzyme may be able to complement E. coli ung mutants. In order to test this hypothesis, the human uracil-DNA glycosylase cDNA was established in a bacterial expression vector. Expression of the human enzyme as a LacZ alpha-humUNG fusion protein was then studied in E. coli ung mutants. E. coli cells lacking uracil-DNA glycosylase activity exhibit a weak mutator phenotype and they are permissive for growth of phages with uracil-containing DNA. Here we show that the expression of human uracil-DNA glycosylase in E. coli can restore the wild type phenotype of ung mutants. These results demonstrate that the evolutionary conservation of the uracil-DNA glycosylase structure is also reflected in the conservation of the mechanism for removal of uracil from DNA.

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Selected References

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  1. Aasland R., Olsen L. C., Spurr N. K., Krokan H. E., Helland D. E. Chromosomal assignment of human uracil-DNA glycosylase to chromosome 12. Genomics. 1990 May;7(1):139–141. doi: 10.1016/0888-7543(90)90532-y. [DOI] [PubMed] [Google Scholar]
  2. Anderson C. T., Friedberg E. C. The presence of nuclear and mitochondrial uracil-DNA glycosylase in extracts of human KB cells. Nucleic Acids Res. 1980 Feb 25;8(4):875–888. [PMC free article] [PubMed] [Google Scholar]
  3. Caradonna S. J., Cheng Y. C. Uracil DNA-glycosylase. Purification and properties of this enzyme isolated from blast cells of acute myelocytic leukemia patients. J Biol Chem. 1980 Mar 25;255(6):2293–2300. [PubMed] [Google Scholar]
  4. Chee M. S., Bankier A. T., Beck S., Bohni R., Brown C. M., Cerny R., Horsnell T., Hutchison C. A., 3rd, Kouzarides T., Martignetti J. A. Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Curr Top Microbiol Immunol. 1990;154:125–169. doi: 10.1007/978-3-642-74980-3_6. [DOI] [PubMed] [Google Scholar]
  5. Doetsch P. W., Cunningham R. P. The enzymology of apurinic/apyrimidinic endonucleases. Mutat Res. 1990 Sep-Nov;236(2-3):173–201. doi: 10.1016/0921-8777(90)90004-o. [DOI] [PubMed] [Google Scholar]
  6. Domena J. D., Mosbaugh D. W. Purification of nuclear and mitochondrial uracil-DNA glycosylase from rat liver. Identification of two distinct subcellular forms. Biochemistry. 1985 Dec 3;24(25):7320–7328. doi: 10.1021/bi00346a045. [DOI] [PubMed] [Google Scholar]
  7. Domena J. D., Timmer R. T., Dicharry S. A., Mosbaugh D. W. Purification and properties of mitochondrial uracil-DNA glycosylase from rat liver. Biochemistry. 1988 Sep 6;27(18):6742–6751. doi: 10.1021/bi00418a015. [DOI] [PubMed] [Google Scholar]
  8. Dower W. J., Miller J. F., Ragsdale C. W. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. doi: 10.1093/nar/16.13.6127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Duncan B. K., Rockstroh P. A., Warner H. R. Escherichia coli K-12 mutants deficient in uracil-DNA glycosylase. J Bacteriol. 1978 Jun;134(3):1039–1045. doi: 10.1128/jb.134.3.1039-1045.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Karran P., Cone R., Friedberg E. C. Specificity of the bacteriophage PBS2 induced inhibitor of uracil-DNA glycosylase. Biochemistry. 1981 Oct 13;20(21):6092–6096. doi: 10.1021/bi00524a027. [DOI] [PubMed] [Google Scholar]
  11. Krokan H., Wittwer C. U. Uracil DNa-glycosylase from HeLa cells: general properties, substrate specificity and effect of uracil analogs. Nucleic Acids Res. 1981 Jun 11;9(11):2599–2613. doi: 10.1093/nar/9.11.2599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. Lindahl T. An N-glycosidase from Escherichia coli that releases free uracil from DNA containing deaminated cytosine residues. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3649–3653. doi: 10.1073/pnas.71.9.3649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Lindahl T., Nyberg B. Heat-induced deamination of cytosine residues in deoxyribonucleic acid. Biochemistry. 1974 Jul 30;13(16):3405–3410. doi: 10.1021/bi00713a035. [DOI] [PubMed] [Google Scholar]
  18. Mullaney J., Moss H. W., McGeoch D. J. Gene UL2 of herpes simplex virus type 1 encodes a uracil-DNA glycosylase. J Gen Virol. 1989 Feb;70(Pt 2):449–454. doi: 10.1099/0022-1317-70-2-449. [DOI] [PubMed] [Google Scholar]
  19. Muller S. J., Caradonna S. Isolation and characterization of a human cDNA encoding uracil-DNA glycosylase. Biochim Biophys Acta. 1991 Feb 16;1088(2):197–207. doi: 10.1016/0167-4781(91)90055-q. [DOI] [PubMed] [Google Scholar]
  20. Méjean V., Rives I., Claverys J. P. Nucleotide sequence of the Streptococcus pneumoniae ung gene encoding uracil-DNA glycosylase. Nucleic Acids Res. 1990 Nov 25;18(22):6693–6693. doi: 10.1093/nar/18.22.6693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. O'Connor T. R., Laval F. Isolation and structure of a cDNA expressing a mammalian 3-methyladenine-DNA glycosylase. EMBO J. 1990 Oct;9(10):3337–3342. doi: 10.1002/j.1460-2075.1990.tb07534.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ohnishi T., Yuba S., Date T., Utsumi H., Matsukage A. Rat DNA polymerase beta gene can join in excision repair of Escherichia coli. Nucleic Acids Res. 1990 Oct 11;18(19):5673–5676. doi: 10.1093/nar/18.19.5673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Olsen L. C., Aasland R., Wittwer C. U., Krokan H. E., Helland D. E. Molecular cloning of human uracil-DNA glycosylase, a highly conserved DNA repair enzyme. EMBO J. 1989 Oct;8(10):3121–3125. doi: 10.1002/j.1460-2075.1989.tb08464.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Percival K. J., Klein M. B., Burgers P. M. Molecular cloning and primary structure of the uracil-DNA-glycosylase gene from Saccharomyces cerevisiae. J Biol Chem. 1989 Feb 15;264(5):2593–2598. [PubMed] [Google Scholar]
  25. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  26. Seal G., Arenaz P., Sirover M. A. Purification and properties of the human placental uracil DNA glycosylase. Biochim Biophys Acta. 1987 Aug 13;925(2):226–233. doi: 10.1016/0304-4165(87)90113-9. [DOI] [PubMed] [Google Scholar]
  27. Seal G., Sirover M. A. Physical association of the human base-excision repair enzyme uracil DNA glycosylase with the 70,000-dalton catalytic subunit of DNA polymerase alpha. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7608–7612. doi: 10.1073/pnas.83.20.7608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tano K., Shiota S., Collier J., Foote R. S., Mitra S. Isolation and structural characterization of a cDNA clone encoding the human DNA repair protein for O6-alkylguanine. Proc Natl Acad Sci U S A. 1990 Jan;87(2):686–690. doi: 10.1073/pnas.87.2.686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tsurimoto T., Melendy T., Stillman B. Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin. Nature. 1990 Aug 9;346(6284):534–539. doi: 10.1038/346534a0. [DOI] [PubMed] [Google Scholar]
  30. Varshney U., Hutcheon T., van de Sande J. H. Sequence analysis, expression, and conservation of Escherichia coli uracil DNA glycosylase and its gene (ung). J Biol Chem. 1988 Jun 5;263(16):7776–7784. [PubMed] [Google Scholar]
  31. Vollberg T. M., Siegler K. M., Cool B. L., Sirover M. A. Isolation and characterization of the human uracil DNA glycosylase gene. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8693–8697. doi: 10.1073/pnas.86.22.8693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wang Z., Mosbaugh D. W. Uracil-DNA glycosylase inhibitor gene of bacteriophage PBS2 encodes a binding protein specific for uracil-DNA glycosylase. J Biol Chem. 1989 Jan 15;264(2):1163–1171. [PubMed] [Google Scholar]
  33. Warner H. R., Thompson R. B., Mozer T. J., Duncan B. K. The properties of a bacteriophage T5 mutant unable to induce deoxyuridine 5'-triphosphate nucleotidohydrolase. Synthesis of uracil-containing T5 deoxyribonucleic acid. J Biol Chem. 1979 Aug 25;254(16):7534–7539. [PubMed] [Google Scholar]
  34. Wittwer C. U., Bauw G., Krokan H. E. Purification and determination of the NH2-terminal amino acid sequence of uracil-DNA glycosylase from human placenta. Biochemistry. 1989 Jan 24;28(2):780–784. doi: 10.1021/bi00428a055. [DOI] [PubMed] [Google Scholar]
  35. Wittwer C. U., Krokan H. Uracil-DNA glycosylase in HeLa S3 cells: interconvertibility of 50 and 20 kDa forms and similarity of the nuclear and mitochondrial form of the enzyme. Biochim Biophys Acta. 1985 Dec 20;832(3):308–318. doi: 10.1016/0167-4838(85)90264-x. [DOI] [PubMed] [Google Scholar]
  36. Worrad D. M., Caradonna S. Identification of the coding sequence for herpes simplex virus uracil-DNA glycosylase. J Virol. 1988 Dec;62(12):4774–4777. doi: 10.1128/jvi.62.12.4774-4777.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

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