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. 1992 Nov;12(11):5159–5173. doi: 10.1128/mcb.12.11.5159

C-terminal truncation of RAP1 results in the deregulation of telomere size, stability, and function in Saccharomyces cerevisiae.

G Kyrion 1, K A Boakye 1, A J Lustig 1
PMCID: PMC360450  PMID: 1406688

Abstract

The Saccharomyces cerevisiae DNA-binding protein RAP1 is capable of binding in vitro to sequences from a wide variety of genomic loci, including upstream activating sequence elements, the HML and HMR silencer regions, and the poly(G1-3T) tracts of telomeres. Recent biochemical and genetic studies have suggested that RAP1 physically and functionally interacts with the yeast telomere. To further investigate the role of RAP1 at the telomere, we have identified and characterized three intragenic suppressors of a temperature-sensitive allele of RAP1, rap1-5. These telomere deficiency (rap1t) alleles confer several novel phenotypes. First, telomere tract size elongates to up to 4 kb greater than sizes of wild-type or rap1-5 telomeres. Second, telomeres are highly unstable and are subject to rapid, but reversible, deletion of part or all of the increase in telomeric tract length. Telomeric deletion does not require the RAD52 or RAD1 gene product. Third, chromosome loss and nondisjunction rates are elevated 15- to 30-fold above wild-type levels. Sequencing analysis has shown that each rap1t allele contains a nonsense mutation within a discrete region between amino acids 663 and 684. Mobility shift and Western immunoblot analyses indicate that each allele produces a truncated RAP1 protein, lacking the C-terminal 144 to 165 amino acids but capable of efficient DNA binding. These data suggest that RAP1 is a central regulator of both telomere and chromosome stability and define a C-terminal domain that, while dispensable for viability, is required for these telomeric functions.

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Selected References

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

  1. Blackburn E. H., Chiou S. S. Non-nucleosomal packaging of a tandemly repeated DNA sequence at termini of extrachromosomal DNA coding for rRNA in Tetrahymena. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2263–2267. doi: 10.1073/pnas.78.4.2263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boeke J. D., Trueheart J., Natsoulis G., Fink G. R. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 1987;154:164–175. doi: 10.1016/0076-6879(87)54076-9. [DOI] [PubMed] [Google Scholar]
  3. Buchman A. R., Lue N. F., Kornberg R. D. Connections between transcriptional activators, silencers, and telomeres as revealed by functional analysis of a yeast DNA-binding protein. Mol Cell Biol. 1988 Dec;8(12):5086–5099. doi: 10.1128/mcb.8.12.5086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Budarf M. L., Blackburn E. H. Chromatin structure of the telomeric region and 3'-nontranscribed spacer of Tetrahymena ribosomal RNA genes. J Biol Chem. 1986 Jan 5;261(1):363–369. [PubMed] [Google Scholar]
  5. Carson M. J., Hartwell L. CDC17: an essential gene that prevents telomere elongation in yeast. Cell. 1985 Aug;42(1):249–257. doi: 10.1016/s0092-8674(85)80120-3. [DOI] [PubMed] [Google Scholar]
  6. Chan C. S., Tye B. K. Organization of DNA sequences and replication origins at yeast telomeres. Cell. 1983 Jun;33(2):563–573. doi: 10.1016/0092-8674(83)90437-3. [DOI] [PubMed] [Google Scholar]
  7. Conrad M. N., Wright J. H., Wolf A. J., Zakian V. A. RAP1 protein interacts with yeast telomeres in vivo: overproduction alters telomere structure and decreases chromosome stability. Cell. 1990 Nov 16;63(4):739–750. doi: 10.1016/0092-8674(90)90140-a. [DOI] [PubMed] [Google Scholar]
  8. Coren J. S., Epstein E. M., Vogt V. M. Characterization of a telomere-binding protein from Physarum polycephalum. Mol Cell Biol. 1991 Apr;11(4):2282–2290. doi: 10.1128/mcb.11.4.2282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Giesman D., Best L., Tatchell K. The role of RAP1 in the regulation of the MAT alpha locus. Mol Cell Biol. 1991 Feb;11(2):1069–1079. doi: 10.1128/mcb.11.2.1069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gottschling D. E., Aparicio O. M., Billington B. L., Zakian V. A. Position effect at S. cerevisiae telomeres: reversible repression of Pol II transcription. Cell. 1990 Nov 16;63(4):751–762. doi: 10.1016/0092-8674(90)90141-z. [DOI] [PubMed] [Google Scholar]
  11. Gottschling D. E., Cech T. R. Chromatin structure of the molecular ends of Oxytricha macronuclear DNA: phased nucleosomes and a telomeric complex. Cell. 1984 Sep;38(2):501–510. doi: 10.1016/0092-8674(84)90505-1. [DOI] [PubMed] [Google Scholar]
  12. Gottschling D. E., Zakian V. A. Telomere proteins: specific recognition and protection of the natural termini of Oxytricha macronuclear DNA. Cell. 1986 Oct 24;47(2):195–205. doi: 10.1016/0092-8674(86)90442-3. [DOI] [PubMed] [Google Scholar]
  13. Greider C. W., Blackburn E. H. A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nature. 1989 Jan 26;337(6205):331–337. doi: 10.1038/337331a0. [DOI] [PubMed] [Google Scholar]
  14. Greider C. W., Blackburn E. H. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell. 1985 Dec;43(2 Pt 1):405–413. doi: 10.1016/0092-8674(85)90170-9. [DOI] [PubMed] [Google Scholar]
  15. Greider C. W., Blackburn E. H. The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity. Cell. 1987 Dec 24;51(6):887–898. doi: 10.1016/0092-8674(87)90576-9. [DOI] [PubMed] [Google Scholar]
  16. Gualberto A., Patrick R. M., Walsh K. Nucleic acid specificity of a vertebrate telomere-binding protein: evidence for G-G base pair recognition at the core-binding site. Genes Dev. 1992 May;6(5):815–824. doi: 10.1101/gad.6.5.815. [DOI] [PubMed] [Google Scholar]
  17. Hardy C. F., Balderes D., Shore D. Dissection of a carboxy-terminal region of the yeast regulatory protein RAP1 with effects on both transcriptional activation and silencing. Mol Cell Biol. 1992 Mar;12(3):1209–1217. doi: 10.1128/mcb.12.3.1209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hardy C. F., Sussel L., Shore D. A RAP1-interacting protein involved in transcriptional silencing and telomere length regulation. Genes Dev. 1992 May;6(5):801–814. doi: 10.1101/gad.6.5.801. [DOI] [PubMed] [Google Scholar]
  19. Hegemann J. H., Shero J. H., Cottarel G., Philippsen P., Hieter P. Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae. Mol Cell Biol. 1988 Jun;8(6):2523–2535. doi: 10.1128/mcb.8.6.2523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Henry Y. A., Chambers A., Tsang J. S., Kingsman A. J., Kingsman S. M. Characterisation of the DNA binding domain of the yeast RAP1 protein. Nucleic Acids Res. 1990 May 11;18(9):2617–2623. doi: 10.1093/nar/18.9.2617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hieter P., Mann C., Snyder M., Davis R. W. Mitotic stability of yeast chromosomes: a colony color assay that measures nondisjunction and chromosome loss. Cell. 1985 Feb;40(2):381–392. doi: 10.1016/0092-8674(85)90152-7. [DOI] [PubMed] [Google Scholar]
  22. Hofmann J. F., Laroche T., Brand A. H., Gasser S. M. RAP-1 factor is necessary for DNA loop formation in vitro at the silent mating type locus HML. Cell. 1989 Jun 2;57(5):725–737. doi: 10.1016/0092-8674(89)90788-5. [DOI] [PubMed] [Google Scholar]
  23. Kanik-Ennulat C., Neff N. Vanadate-resistant mutants of Saccharomyces cerevisiae show alterations in protein phosphorylation and growth control. Mol Cell Biol. 1990 Mar;10(3):898–909. doi: 10.1128/mcb.10.3.898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Klein H. L. Different types of recombination events are controlled by the RAD1 and RAD52 genes of Saccharomyces cerevisiae. Genetics. 1988 Oct;120(2):367–377. doi: 10.1093/genetics/120.2.367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kurtz S., Shore D. RAP1 protein activates and silences transcription of mating-type genes in yeast. Genes Dev. 1991 Apr;5(4):616–628. doi: 10.1101/gad.5.4.616. [DOI] [PubMed] [Google Scholar]
  26. Liu Z. P., Tye B. K. A yeast protein that binds to vertebrate telomeres and conserved yeast telomeric junctions. Genes Dev. 1991 Jan;5(1):49–59. doi: 10.1101/gad.5.1.49. [DOI] [PubMed] [Google Scholar]
  27. Longtine M. S., Wilson N. M., Petracek M. E., Berman J. A yeast telomere binding activity binds to two related telomere sequence motifs and is indistinguishable from RAP1. Curr Genet. 1989 Oct;16(4):225–239. doi: 10.1007/BF00422108. [DOI] [PubMed] [Google Scholar]
  28. Lundblad V., Szostak J. W. A mutant with a defect in telomere elongation leads to senescence in yeast. Cell. 1989 May 19;57(4):633–643. doi: 10.1016/0092-8674(89)90132-3. [DOI] [PubMed] [Google Scholar]
  29. Lustig A. J., Kurtz S., Shore D. Involvement of the silencer and UAS binding protein RAP1 in regulation of telomere length. Science. 1990 Oct 26;250(4980):549–553. doi: 10.1126/science.2237406. [DOI] [PubMed] [Google Scholar]
  30. Lustig A. J., Petes T. D. Identification of yeast mutants with altered telomere structure. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1398–1402. doi: 10.1073/pnas.83.5.1398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Morin G. B. The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats. Cell. 1989 Nov 3;59(3):521–529. doi: 10.1016/0092-8674(89)90035-4. [DOI] [PubMed] [Google Scholar]
  32. Murray A. W., Schultes N. P., Szostak J. W. Chromosome length controls mitotic chromosome segregation in yeast. Cell. 1986 May 23;45(4):529–536. doi: 10.1016/0092-8674(86)90284-9. [DOI] [PubMed] [Google Scholar]
  33. Oliver S. G., van der Aart Q. J., Agostoni-Carbone M. L., Aigle M., Alberghina L., Alexandraki D., Antoine G., Anwar R., Ballesta J. P., Benit P. The complete DNA sequence of yeast chromosome III. Nature. 1992 May 7;357(6373):38–46. doi: 10.1038/357038a0. [DOI] [PubMed] [Google Scholar]
  34. Ozenberger B. A., Roeder G. S. A unique pathway of double-strand break repair operates in tandemly repeated genes. Mol Cell Biol. 1991 Mar;11(3):1222–1231. doi: 10.1128/mcb.11.3.1222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Price C. M., Cech T. R. Telomeric DNA-protein interactions of Oxytricha macronuclear DNA. Genes Dev. 1987 Oct;1(8):783–793. doi: 10.1101/gad.1.8.783. [DOI] [PubMed] [Google Scholar]
  36. Price C. M. Telomere structure in Euplotes crassus: characterization of DNA-protein interactions and isolation of a telomere-binding protein. Mol Cell Biol. 1990 Jul;10(7):3421–3431. doi: 10.1128/mcb.10.7.3421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Raghuraman M. K., Cech T. R. Assembly and self-association of oxytricha telomeric nucleoprotein complexes. Cell. 1989 Nov 17;59(4):719–728. doi: 10.1016/0092-8674(89)90018-4. [DOI] [PubMed] [Google Scholar]
  38. Raghuraman M. K., Dunn C. J., Hicke B. J., Cech T. R. Oxytricha telomeric nucleoprotein complexes reconstituted with synthetic DNA. Nucleic Acids Res. 1989 Jun 12;17(11):4235–4253. doi: 10.1093/nar/17.11.4235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shampay J., Blackburn E. H. Generation of telomere-length heterogeneity in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1988 Jan;85(2):534–538. doi: 10.1073/pnas.85.2.534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Shampay J., Szostak J. W., Blackburn E. H. DNA sequences of telomeres maintained in yeast. Nature. 1984 Jul 12;310(5973):154–157. doi: 10.1038/310154a0. [DOI] [PubMed] [Google Scholar]
  41. Shippen-Lentz D., Blackburn E. H. Functional evidence for an RNA template in telomerase. Science. 1990 Feb 2;247(4942):546–552. doi: 10.1126/science.1689074. [DOI] [PubMed] [Google Scholar]
  42. Shore D., Nasmyth K. Purification and cloning of a DNA binding protein from yeast that binds to both silencer and activator elements. Cell. 1987 Dec 4;51(5):721–732. doi: 10.1016/0092-8674(87)90095-x. [DOI] [PubMed] [Google Scholar]
  43. Shore D., Stillman D. J., Brand A. H., Nasmyth K. A. Identification of silencer binding proteins from yeast: possible roles in SIR control and DNA replication. EMBO J. 1987 Feb;6(2):461–467. doi: 10.1002/j.1460-2075.1987.tb04776.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sussel L., Shore D. Separation of transcriptional activation and silencing functions of the RAP1-encoded repressor/activator protein 1: isolation of viable mutants affecting both silencing and telomere length. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7749–7753. doi: 10.1073/pnas.88.17.7749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Szostak J. W., Blackburn E. H. Cloning yeast telomeres on linear plasmid vectors. Cell. 1982 May;29(1):245–255. doi: 10.1016/0092-8674(82)90109-x. [DOI] [PubMed] [Google Scholar]
  47. Thomas B. J., Rothstein R. The genetic control of direct-repeat recombination in Saccharomyces: the effect of rad52 and rad1 on mitotic recombination at GAL10, a transcriptionally regulated gene. Genetics. 1989 Dec;123(4):725–738. doi: 10.1093/genetics/123.4.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Walmsley R. M., Petes T. D. Genetic control of chromosome length in yeast. Proc Natl Acad Sci U S A. 1985 Jan;82(2):506–510. doi: 10.1073/pnas.82.2.506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wright J. H., Gottschling D. E., Zakian V. A. Saccharomyces telomeres assume a non-nucleosomal chromatin structure. Genes Dev. 1992 Feb;6(2):197–210. doi: 10.1101/gad.6.2.197. [DOI] [PubMed] [Google Scholar]
  50. Zahler A. M., Prescott D. M. Telomere terminal transferase activity in the hypotrichous ciliate Oxytricha nova and a model for replication of the ends of linear DNA molecules. Nucleic Acids Res. 1988 Jul 25;16(14B):6953–6972. doi: 10.1093/nar/16.14.6953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zakian V. A. Structure and function of telomeres. Annu Rev Genet. 1989;23:579–604. doi: 10.1146/annurev.ge.23.120189.003051. [DOI] [PubMed] [Google Scholar]
  52. de Lange T. Human telomeres are attached to the nuclear matrix. EMBO J. 1992 Feb;11(2):717–724. doi: 10.1002/j.1460-2075.1992.tb05104.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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