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. 1993 Dec;13(12):7757–7765. doi: 10.1128/mcb.13.12.7757

The Saccharomyces cerevisiae DNA repair gene RAD23 encodes a nuclear protein containing a ubiquitin-like domain required for biological function.

J F Watkins 1, P Sung 1, L Prakash 1, S Prakash 1
PMCID: PMC364847  PMID: 8246991

Abstract

In eukaryotes, the posttranslational conjugation of ubiquitin to various cellular proteins marks them for degradation. Interestingly, several proteins have been reported to contain ubiquitin-like (ub-like) domains that are in fact specified by the DNA coding sequences of the proteins. The biological role of the ub-like domain in these proteins is not known; however, it has been proposed that this domain functions as a degradation signal rendering the proteins unstable. Here, we report that the product of the Saccharomyces cerevisiae RAD23 gene, which is involved in excision repair of UV-damaged DNA, bears a ub-like domain at its amino terminus. This finding has presented an opportunity to define the functional significance of this domain. We show that deletion of the ub-like domain impairs the DNA repair function of RAD23 and that this domain can be functionally substituted by the authentic ubiquitin sequence. Surprisingly, RAD23 is highly stable, and the studies reported herein indicate that its ub-like domain does not mediate protein degradation. Thus, in RAD23 at least, the ub-like domain affects protein function in a nonproteolytic manner.

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Selected References

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  1. Alani E., Cao L., Kleckner N. A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains. Genetics. 1987 Aug;116(4):541–545. doi: 10.1534/genetics.112.541.test. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Banerji J., Sands J., Strominger J. L., Spies T. A gene pair from the human major histocompatibility complex encodes large proline-rich proteins with multiple repeated motifs and a single ubiquitin-like domain. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2374–2378. doi: 10.1073/pnas.87.6.2374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bang D. D., Verhage R., Goosen N., Brouwer J., van de Putte P. Molecular cloning of RAD16, a gene involved in differential repair in Saccharomyces cerevisiae. Nucleic Acids Res. 1992 Aug 11;20(15):3925–3931. doi: 10.1093/nar/20.15.3925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bankmann M., Prakash L., Prakash S. Yeast RAD14 and human xeroderma pigmentosum group A DNA-repair genes encode homologous proteins. Nature. 1992 Feb 6;355(6360):555–558. doi: 10.1038/355555a0. [DOI] [PubMed] [Google Scholar]
  5. Chau V., Tobias J. W., Bachmair A., Marriott D., Ecker D. J., Gonda D. K., Varshavsky A. A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science. 1989 Mar 24;243(4898):1576–1583. doi: 10.1126/science.2538923. [DOI] [PubMed] [Google Scholar]
  6. Ecker D. J., Khan M. I., Marsh J., Butt T. R., Crooke S. T. Chemical synthesis and expression of a cassette adapted ubiquitin gene. J Biol Chem. 1987 Mar 15;262(8):3524–3527. [PubMed] [Google Scholar]
  7. Filippi M., Tribioli C., Toniolo D. Linkage and sequence conservation of the X-linked genes DXS253E (P3) and DXS254E (GdX) in mouse and man. Genomics. 1990 Jul;7(3):453–457. doi: 10.1016/0888-7543(90)90184-v. [DOI] [PubMed] [Google Scholar]
  8. Finley D., Bartel B., Varshavsky A. The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis. Nature. 1989 Mar 30;338(6214):394–401. doi: 10.1038/338394a0. [DOI] [PubMed] [Google Scholar]
  9. Finley D., Chau V. Ubiquitination. Annu Rev Cell Biol. 1991;7:25–69. doi: 10.1146/annurev.cb.07.110191.000325. [DOI] [PubMed] [Google Scholar]
  10. Gietz R. D., Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988 Dec 30;74(2):527–534. doi: 10.1016/0378-1119(88)90185-0. [DOI] [PubMed] [Google Scholar]
  11. Gribskov M., Burgess R. R. Sigma factors from E. coli, B. subtilis, phage SP01, and phage T4 are homologous proteins. Nucleic Acids Res. 1986 Aug 26;14(16):6745–6763. doi: 10.1093/nar/14.16.6745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Haas A. L., Bright P. M. The immunochemical detection and quantitation of intracellular ubiquitin-protein conjugates. J Biol Chem. 1985 Oct 15;260(23):12464–12473. [PubMed] [Google Scholar]
  13. Hershko A. The ubiquitin pathway for protein degradation. Trends Biochem Sci. 1991 Jul;16(7):265–268. doi: 10.1016/0968-0004(91)90101-z. [DOI] [PubMed] [Google Scholar]
  14. Hochstrasser M., Ellison M. J., Chau V., Varshavsky A. The short-lived MAT alpha 2 transcriptional regulator is ubiquitinated in vivo. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4606–4610. doi: 10.1073/pnas.88.11.4606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Johnson E. S., Bartel B., Seufert W., Varshavsky A. Ubiquitin as a degradation signal. EMBO J. 1992 Feb;11(2):497–505. doi: 10.1002/j.1460-2075.1992.tb05080.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Müller-Taubenberger A., Westphal M., Jaeger E., Noegel A., Gerisch G. Complete cDNA sequence of a Dictyostelium ubiquitin with a carboxy-terminal tail and identification of the protein using an anti-peptide antibody. FEBS Lett. 1988 Mar 14;229(2):273–278. doi: 10.1016/0014-5793(88)81139-6. [DOI] [PubMed] [Google Scholar]
  18. Neves A. M., Barahona I., Galego L., Rodrigues-Pousada C. Ubiquitin genes in Tetrahymena pyriformis and their expression during heat shock. Gene. 1988 Dec 15;73(1):87–96. doi: 10.1016/0378-1119(88)90315-0. [DOI] [PubMed] [Google Scholar]
  19. Neves A., Guerreiro P., Rodrigues-Pousada C. Striking changes in polyubiquitin genes of Tetrahymena pyriformis. Nucleic Acids Res. 1990 Feb 11;18(3):656–656. doi: 10.1093/nar/18.3.656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ozkaynak E., Finley D., Solomon M. J., Varshavsky A. The yeast ubiquitin genes: a family of natural gene fusions. EMBO J. 1987 May;6(5):1429–1439. doi: 10.1002/j.1460-2075.1987.tb02384.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Park E., Guzder S. N., Koken M. H., Jaspers-Dekker I., Weeda G., Hoeijmakers J. H., Prakash S., Prakash L. RAD25 (SSL2), the yeast homolog of the human xeroderma pigmentosum group B DNA repair gene, is essential for viability. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11416–11420. doi: 10.1073/pnas.89.23.11416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pringle J. R., Adams A. E., Drubin D. G., Haarer B. K. Immunofluorescence methods for yeast. Methods Enzymol. 1991;194:565–602. doi: 10.1016/0076-6879(91)94043-c. [DOI] [PubMed] [Google Scholar]
  23. Rechid R., Vingron M., Argos P. A new interactive protein sequence alignment program and comparison of its results with widely used algorithms. Comput Appl Biosci. 1989 Apr;5(2):107–113. doi: 10.1093/bioinformatics/5.2.107. [DOI] [PubMed] [Google Scholar]
  24. Reich N., Evans B., Levy D., Fahey D., Knight E., Jr, Darnell J. E., Jr Interferon-induced transcription of a gene encoding a 15-kDa protein depends on an upstream enhancer element. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6394–6398. doi: 10.1073/pnas.84.18.6394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Smerdon M. J., Thoma F. Site-specific DNA repair at the nucleosome level in a yeast minichromosome. Cell. 1990 May 18;61(4):675–684. doi: 10.1016/0092-8674(90)90479-x. [DOI] [PubMed] [Google Scholar]
  27. Swindle J., Ajioka J., Eisen H., Sanwal B., Jacquemot C., Browder Z., Buck G. The genomic organization and transcription of the ubiquitin genes of Trypanosoma cruzi. EMBO J. 1988 Apr;7(4):1121–1127. doi: 10.1002/j.1460-2075.1988.tb02921.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Toniolo D., Persico M., Alcalay M. A "housekeeping" gene on the X chromosome encodes a protein similar to ubiquitin. Proc Natl Acad Sci U S A. 1988 Feb;85(3):851–855. doi: 10.1073/pnas.85.3.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Watkins J. F., Sung P., Prakash S., Prakash L. The extremely conserved amino terminus of RAD6 ubiquitin-conjugating enzyme is essential for amino-end rule-dependent protein degradation. Genes Dev. 1993 Feb;7(2):250–261. doi: 10.1101/gad.7.2.250. [DOI] [PubMed] [Google Scholar]
  31. Wiborg O., Pedersen M. S., Wind A., Berglund L. E., Marcker K. A., Vuust J. The human ubiquitin multigene family: some genes contain multiple directly repeated ubiquitin coding sequences. EMBO J. 1985 Mar;4(3):755–759. doi: 10.1002/j.1460-2075.1985.tb03693.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. doi: 10.1089/dna.1.1984.3.479. [DOI] [PubMed] [Google Scholar]

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