1887

Abstract

Uracil, a promutagenic base, appears in DNA either by deamination of cytosine or by incorporation of dUMP by DNA polymerases. This unconventional base in DNA is removed by uracil-DNA glycosylase (UDG). Interestingly, a bacteriophage-encoded short polypeptide, UDG inhibitor (Ugi), specifically inhibits UDGs by forming a tight complex. Three-dimensional structures of the complexes of Ugi with UDGs from , human and herpes simplex virus have shown that two of the structural elements in Ugi, the hydrophobic pocket and the 1-edge, establish key interactions with UDGs. In this report the characterization of complexes of Ugi with UDGs from , a pathogenic bacterium, and , a widely used model organism for the former, is described. Unlike the () UDG-Ugi complex, which is stable to treatment with 8 M urea, the mycobacterial UDG-Ugi complexes dissociate in 5–6 M urea. Furthermore, the Ugi from the complexes of mycobacterial UDGs can be exchanged by the DNA substrate. Interestingly, while UDG sequestered Ugi into the UDG-Ugi complex when incubated with mycobacterial UDG-Ugi complexes, even a large excess of mycobacterial UDGs failed to sequester Ugi from the UDG-Ugi complex. However, the () UDG-Ugi complex was seen when UDG was incubated with () UDG-Ugi or UDG(L191G)-Ugi complexes. The reversible nature of the complexes of Ugi with mycobacterial UDGs (which naturally lack some of the structural elements important for interaction with the 1-edge of Ugi) and with mutants of UDG (which are deficient in interaction with the hydrophobic pocket of Ugi) highlights the significance of both classes of interaction in formation of UDG-Ugi complexes. Furthermore, it is shown that even though mycobacterial UDG-Ugi complexes dissociate in 5–6 M urea, Ugi is still a potent inhibitor of UDG activity in mycobacteria.

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2003-07-01
2024-03-28
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References

  1. Acharya N., Varshney U. 2002; Biochemical properties of single stranded DNA binding protein from Mycobacterium smegmatis , a fast growing mycobacterium and its physical and functional interaction with uracil DNA glycosylases. J Mol Biol 318:1251–1264
    [Google Scholar]
  2. Acharya N., Roy S., Varshney U. 2002; Mutational analysis of the uracil DNA glycosylase inhibitor protein and its interaction with Escherichia coli uracil DNA glycosylase. J Mol Biol 321:579–590
    [Google Scholar]
  3. Aravind L., Koonin E. V. 2000; The α / β fold uracil DNA glycosylases: a common origin with diverse fates. Genome Biol 1:1–8
    [Google Scholar]
  4. Bennett S. E., Mosbaugh D. W. 1992; Characterization of the Escherichia coli uracil-DNA glycosylase inhibitor protein complex. J Biol Chem 267:22512–22521
    [Google Scholar]
  5. Bennett S. E., Schimerlik M. I., Mosbaugh D. W. 1993; Kinetics of the uracil-DNA glycosylase/inhibitor protein association. Ung interaction with Ugi, nucleic acids, and uracil compounds. J Biol Chem 268:26879–26885
    [Google Scholar]
  6. Chen J. D., Lacks S. A. 1991; Role of uracil-DNA glycosylase in mutation avoidance by Streptococcus pneumoniae . J Bacteriol 173:283–290
    [Google Scholar]
  7. Cole S. T., Brosch R., Parkhill J. 39 other authors 1998; Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537–544
    [Google Scholar]
  8. Cone R., Bonura T., Friedberg E. C. 1980; Inhibitor of uracil-DNA glycosylase induced by bacteriophage PBS2. Purification and preliminary characterization. J Biol Chem 255:10354–10358
    [Google Scholar]
  9. Duncan B. K., Weiss B. 1982; Specific mutator effects of ung (uracil-DNA glycosylase) mutations in Escherichia coli . J Bacteriol 151:750–755
    [Google Scholar]
  10. Handa P., Roy S., Varshney U. 2001; The role of leucine 191 of Escherichia coli in forming a stable complex with a substrate mimic, Ugi, and in uracil excision from synthetic substrates. J Biol Chem 276:17324–17331
    [Google Scholar]
  11. Handa P., Acharya N., Talawar R. K., Roy S., Varshney U. 2002; Contrasting effects of mutating active site residues, aspartic acid 64 and histidine 187 of Escherichia coli uracil-DNA glycosylase on uracil excision and interaction with an inhibitor protein. Ind J Biochem Biophys 29:312–317
    [Google Scholar]
  12. Impellizzeri K. J., Anderson B., Burgers P. M. 1991; The spectrum of spontaneous mutations in a Saccharomyces cerevisiae uracil-DNA-glycosylase mutant limits the function of this enzyme to cytosine deamination repair. J Bacteriol 21:6807–6810
    [Google Scholar]
  13. Krokan H. E., Standal R., Slupphaug G. 1997; DNA glycosylases in the base excision repair of DNA. Biochem J 325:1–15
    [Google Scholar]
  14. Kumar N. V., Varshney U. 1994; Inefficient excision of uracil from loop regions of DNA oligomers by E. coli uracil-DNA glycosylase. Nucleic Acids Res 18:3737–3741
    [Google Scholar]
  15. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
    [Google Scholar]
  16. Lindahl T. 1974; An N -glycosidase from Escherichia coli that releases free uracil from DNA containing deaminated cytosine residues. Proc Natl Acad Sci U S A 71:3649–3653
    [Google Scholar]
  17. Lundquist A. J., Beger R. D., Bennett S. E., Bolton P. H., Mosbaugh D. W. 1997; Site-directed mutagenesis and characterization of uracil-DNA glycosylase inhibitor protein. Role of specific carboxylic amino acids in complex formation with Escherichia coli uracil-DNA glycosylase. J Biol Chem 272:21408–21419
    [Google Scholar]
  18. Millins A. K., Carpenter M. S., DeLange A. M. 1994; The vaccinia virus-encoded uracil DNA glycosylase has an essential role in viral DNA replication. Virology 198:504–513
    [Google Scholar]
  19. Mol C. D., Arvai A. S., Sanderson R. J., Slupphaug G., Kavli B., Krokan H. E., Mosbaugh D. W., Tainer J. A. 1995; Crystal structure of the human Uracil-DNA glycosylase in complex with a protein inhibitor: protein mimicry of DNA. Cell 82:701–708
    [Google Scholar]
  20. Mullaney J., Moss H. W., McGeoch D. J. 1989; Gene UL2 of herpes simplex virus type 1 encodes a uracil-DNA glycosylase. J Gen Virol 70:449–454
    [Google Scholar]
  21. Nilsen H., Rosewell I., Robins P. 7 other authors 2000; Uracil-DNA glycosylase (UNG)-deficient mice reveal a primary role of the enzyme during DNA replication. Mol Cell 5:1059–1065
    [Google Scholar]
  22. Olsen L. C., Aasland R., Krokan H. E., Helland D. E. 1991; Human uracil-DNA glycosylase complements E. coli ung mutants. Nucleic Acids Res 19:4473–4478
    [Google Scholar]
  23. Purnapatre K., Handa P., Venkatesh J., Varshney U. 1999; Differential effects of single-stranded DNA binding proteins (SSBs) on uracil-DNA glycosylases (UDGs) from Escherichia coli and mycobacteria. Nucleic Acids Res 27:3487–3492
    [Google Scholar]
  24. Putnam C. D., Shroyer M. J. N., Lundquist A. J., Mol C. D., Arvai A. S., Mosbaugh D. W. 1999; Protein mimicry of DNA from crystal structures of the uracil-DNA glycosylase inhibitor protein and its complex with Escherichia coli uracil-DNA glycosylase. J Mol Biol 287:331–346
    [Google Scholar]
  25. Pyles R. B., Thompson R. L. 1994; Evidence that the herpes simplex virus type 1 uracil DNA glycosylase is required for efficient viral replication and latency in the murine nervous system. J Virol 68:4963–4972
    [Google Scholar]
  26. Ravishankar R., Bidya Sagar M., Roy S., Purnapatre K., Handa P., Varshney U., Vijayan M. 1998; X-ray analysis of a complex of E. coli uracil-DNA glycosylase (EcUDG) with its proteinaceous inhibitor. The structure elucidation of a prokaryotic UDG. Nucleic Acids Res 26:4880–4887
    [Google Scholar]
  27. Roy S., Purnapatre K., Handa P., Boyanapalli M., Varshney U. 1998; Use of coupled transcriptional system for consistent overexpression and purification of UDG-Ugi complex and Ugi from E. coli . Protein Expr Purif 13:155–162
    [Google Scholar]
  28. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  29. Sasaki Y., Ishikawa J., Yamashita A., Oshima K., Kenri T., Furuya K., Yoshino C., Horino A., Shiba T., Sasaki T., Hattori M. 2002; The complete genomic sequence of Mycoplasma penetrans , an intracellular bacterial pathogen in humans. Nucleic Acids Res 30:5293–5300
    [Google Scholar]
  30. Savva R., Pearl L. H. 1995; Nucleotide mimicry in the crystal structure of the uracil-DNA glycosylase-uracil glycosylase inhibitor protein complex. Nat Struct Biol 2:752–755
    [Google Scholar]
  31. Sedmak J. J., Grossberg S. E. 1977; A rapid, sensitive, and versatile assay for protein using Coomassie brilliant blue G250. Anal Biochem 79:544–552
    [Google Scholar]
  32. Snapper S. B., Melton R. E., Mustafa S., Kieser T., Jacobs W. R. Jr 1990; Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis . Mol Microbiol 4:1911–1919
    [Google Scholar]
  33. Stuart D. T., Upton C., Higman M. A., Niles E. G., McFadden G. 1993; A poxvirus-encoded uracil DNA glycosylase is essential for virus viability. J Virol 67:2503–2512
    [Google Scholar]
  34. Wang Z., Mosbaugh D. W. 1989; Uracil-DNA glycosylase inhibitor gene of bacteriophage PBS2 encodes a binding protein specific for uracil-DNA glycosylase. J Biol Chem 264:1163–1171
    [Google Scholar]
  35. Willetts K. E., Rey F., Agostini I., Navarro J. M., Baudat Y., Vigne R., Sire J. 1999; DNA repair enzyme uracil DNA glycosylase is specifically incorporated into human immunodeficiency virus type 1 viral particles through a Vpr-independent mechanism. J Virol 73:1682–1688
    [Google Scholar]
  36. Williams M. V., Pollack J. D. 1990; A mollicute (mycoplasma) DNA repair enzyme: purification and characterization of uracil-DNA glycosylase. J Bacteriol 172:2979–2985
    [Google Scholar]
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