DEG1, encoding the tRNA:pseudouridine synthase Pus3p, impacts HOT1-stimulated recombination in Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae, HOT1-stimulated recombination has been implicated in maintaining homology between repeated ribosomal RNA genes. The ability of HOT1 to stimulate genetic exchange requires RNA polymerase I transcription across the recombining sequences. The trans-acting nuclear mutation hrm3-1 specifically reduces HOT1-dependent recombination and prevents cell growth at 37°. The HRM3 gene is identical to DEG1. Excisive, but not gene replacement, recombination is reduced in HOT1-adjacent sequences in deg1Δ mutants. Excisive recombination within the genomic rDNA repeats is also decreased. The hypo-recombination and temperature-sensitive phenotypes of deg1Δ mutants are recessive. Deletion of DEG1 did not affect the rate of transcription from HOT1 or rDNA suggesting that while transcription is necessary it is not sufficient for HOT1 activity. Pseudouridine synthase 3 (Pus3p), the DEG1 gene product, modifies the anticodon arm of transfer RNA at positions 38 and 39 by catalyzing the conversion of uridine to pseudouridine. Cells deficient in pseudouridine synthases encoded by PUS1, PUS2 or PUS4 displayed no recombination defects, indicating that Pus3p plays a specific role in HOT1 activity. Pus3p is unique in its ability to modulate frameshifting and readthrough events during translation, and this aspect of its activity may be responsible for HOT1 recombination phenotypes observed in deg1 mutants.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2

References

  1. Aguilera A, Klein HL (1988) Genetic control of intrachromosomal recombination in Saccharomyces cerevisiae. I. Isolation and genetic characterization of hyper-recombination mutations. Genetics 119:779–790

    PubMed  CAS  Google Scholar 

  2. Amstutz H, Munz P, Heyer WD, Leupoid U, Kohli J (1985) Concerted evolution of tRNA genes: intergenic conversion among three unlinked serine tRNA genes in S. pombe. Cell 40:879–886

    PubMed  Article  CAS  Google Scholar 

  3. Asakura T, Sasaki T, Nagano F, Satoh A, Obaishi H, Nishioka H, Imomura H, Hotta K, Tanaka K, Nakanishi H, Takai Y (1998) Isolation and characterization of a novel actin filament-binding protein from Saccharomyces cerevisiae. Oncogene 16:121–130

    PubMed  Article  CAS  Google Scholar 

  4. Barnes WM (1978) DNA sequence from the histidine operon control region: seven histidine codons in a row. Proc Natl Acad Sci USA 75:4281–4285

    PubMed  Article  CAS  Google Scholar 

  5. Bayev AA, Georgiev OI, Hadjiolov AA, Kermekchiev MB, Nikolaev N, Skryabin KG, Zakharyev VM (1980) The structure of the yeast ribosomal RNA genes. 2. The nucleotide sequence of the initiation site for ribosomal RNA transcription. Nucleic Acids Res 8:4919–4926

    PubMed  Article  CAS  Google Scholar 

  6. Becker HF, Motorin Y, Planta RJ, Grosjean H (1997) The yeast gene YNL292w encodes a pseudouridine synthase (Pus4) catalyzing the formation of psi55 in both mitochondrial and cytoplasmic tRNAs. Nucleic Acids Res 25:4493–4499

    PubMed  Article  CAS  Google Scholar 

  7. Björk GR (1995) Biosynthesis and function of modified nucleosides in tRNA. In: Söll D, RajBhandary UL (eds) tRNA: structure, biosynthesis, and function. ASM Press, Washington, pp 165–205

    Google Scholar 

  8. Brambilla A, Mainieri D, Carbone MLA (1997) A simple signal element mediates transcription termination and mRNA 3′ end formation in the DEG1 gene of Saccharomyces cerevisiae. Mol Gen Genet 254:681–688

    PubMed  Article  CAS  Google Scholar 

  9. Carbone MLA, Solinas M, Sora S, Panzeri L (1991) A gene linked to CEN6 is important for growth of Saccharomyces cerevisiae. Curr Genet 19:1–8

    PubMed  Article  CAS  Google Scholar 

  10. Charette M, Gray MW (2000) Pseudouridine in RNA: what, where, how, and why. IUBMB Life 49:341–351

    PubMed  CAS  Article  Google Scholar 

  11. Defossez PA, Prusty R, Kaeberlein M, Lin SJ, Ferrigno P, Silver PA, Keil RL, Guarente L (1999) Elimination of replication block protein Fob1 extends the life span of yeast mother cells. Mol Cell 3:447–455

    PubMed  Article  CAS  Google Scholar 

  12. Elion EA, Warner JR (1984) The major promoter element of rRNA transcription in yeast lies 2 kb upstream. Cell 39:663–673

    PubMed  Article  CAS  Google Scholar 

  13. Elion EA, Warner JR (1986) An RNA polymerase I enhancer in Saccharomyces cerevisiae. Mol Cell Biol 6:2089–2097

    PubMed  CAS  Google Scholar 

  14. Fiers W, Contreras RR, Haegemann G, Rogiers R, Van de Voorde A, Van Heuverswyn H, Van Herreweghe J, Volckaert G, Ysbaert M (1978) Complete nucleotide sequence of SV40 DNA. Nature 273:113–120

    PubMed  Article  CAS  Google Scholar 

  15. Freedman JA, Jinks-Robertson S (2002) Genetic requirements for spontaneous and transcription-stimulated mitotic recombination in Saccharomyces cerevisiae. Genetics 162:15–27

    PubMed  CAS  Google Scholar 

  16. Gesteland RF, Weiss RB, Atkins JF (1992) Recoding: reprogrammed genetic decoding. Science 257:1640–1641

    PubMed  Article  CAS  Google Scholar 

  17. Gietz RD, Sugino A (1988) New yeast—Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74:527–534

    PubMed  Article  CAS  Google Scholar 

  18. Guldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH (1996) A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 24:2519–2524

    PubMed  Article  CAS  Google Scholar 

  19. Guyer MS (1983) Uses of the transposon γδ in the analysis of cloned genes. Methods Enzymol 101:362–369

    PubMed  CAS  Google Scholar 

  20. Haber JE, Thorburn PC (1984) Healing of broken linear dicentric chromosomes in yeast. Genetics 106:207–226

    PubMed  CAS  Google Scholar 

  21. Hellmuth K, Grosjean H, Motorin Y, Deinert K, Hurt E, Simos G (2000) Cloning and characterization of the Schizosaccharomyces pombe tRNA: pseudouridine synthase Pus1p. Nucleic Acids Res 28:4604–4610

    PubMed  Article  CAS  Google Scholar 

  22. Huang GS, Keil RL (1995) Requirements for activity of the yeast mitotic recombination hotspot HOT1: RNA polymerase I and multiple cis-acting sequences. Genetics 141:845–855

    PubMed  CAS  Google Scholar 

  23. Keil RL, McWilliams AD (1993) A gene with specific and global effects on recombination of sequences from tandemly repeated genes in Saccharomyces cerevisiae. Genetics 135:711–718

    PubMed  CAS  Google Scholar 

  24. Keil RL, Roeder GS (1984) Cis-acting, recombination-stimulating activity in a fragment of the ribosomal DNA of S. cerevisiae. Cell 39:377–386

    PubMed  Article  CAS  Google Scholar 

  25. Klemenz R, Geiduschek EP (1980) The 5′ terminus of the precursor ribosomal RNA of Saccharomyces cerevisiae. Nucleic Acids Res 8:2679–2689

    PubMed  Article  CAS  Google Scholar 

  26. Koonin EV (1996) Pseudouridine synthases: four families of enzymes containing a putative uridine-binding motif also conserved in dUTPases and dCTP deaminases. Nucleic Acids Res 24:2411–2415

    PubMed  Article  CAS  Google Scholar 

  27. Kramer KM, Brock JA, Bloom K, Moore JK, Haber JE (1994) Two different types of double-strand breaks in Saccharomyces cerevisiae are repaired by similar RAD52-independent, nonhomologous recombination events. Mol Cell Biol 14:1293–1301

    PubMed  CAS  Google Scholar 

  28. Lecointe F, Simos G, Sauer A, Hurt EC, Motorin Y, Grosjean H (1998) Characterization of yeast protein Deg1 as pseudouridine synthase (Pus3) catalyzing the formation of psi38 and psi39 in tRNA anticodon loop. J Biol Chem 273:1316–1323

    PubMed  Article  CAS  Google Scholar 

  29. Lecointe F, Namy O, Hatin I, Simos G, Rousset JP, Grosjean H (2002) Lack of pseudouridine 38/39 in the anticodon arm of yeast cytoplasmic tRNA decreases in vivo recoding efficiency. J Biol Chem 277:30445–30453

    PubMed  Article  CAS  Google Scholar 

  30. Lin Y-H, Keil RL (1991) Mutations affecting RNA polymerase I-stimulated exchange and rDNA recombination in yeast. Genetics 127:31–38

    PubMed  CAS  Google Scholar 

  31. Mann C, Davis RW (1983) Instability of dicentric plasmids in yeast. Proc Natl Acad Sci USA 80:228–232

    PubMed  Article  CAS  Google Scholar 

  32. Morris DK, Lundblad V (1997) Programmed translational frameshifting in a gene required for yeast telomere replication. Curr Biol 7:969–976

    PubMed  Article  CAS  Google Scholar 

  33. Motorin Y, Keith G, Simon C, Foiret D, Simos G, Hurt E, Grosjean H (1998) The yeast tRNA: pseudouridine synthase Pus1p displays a multisite substrate specificity. RNA 4:856–869

    PubMed  Article  CAS  Google Scholar 

  34. Namy O, Duchateau-Nguyen G, Rousset JP (2002) Translational readthrough of the PDE2 stop codon modulates cAMP levels in Saccharomyces cerevisiae. Mol Microbiol 43:641–652

    PubMed  Article  CAS  Google Scholar 

  35. Ouspenski S II, Elledge SJ, Brinkley BR (1999) New yeast genes important for chromosome integrity and segregation identified by dosage effects on genome stability. Nucleic Acids Res 27:3001–3008

    PubMed  Article  CAS  Google Scholar 

  36. Pâques F, Haber JE (1999) Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 63:349–404

    PubMed  Google Scholar 

  37. Prusty R, Keil RL (2004) SCH9, a putative protein kinase from Saccharomyces cerevisiae, affects HOT1-stimulated recombination. Mol Gen Genomics 272:264–274

    Article  CAS  Google Scholar 

  38. Raychaudhuri S, Niu L, Conrad J, Lane BG, Ofengand J (1999) Functional effect of deletion and mutation of the Escherichia coli ribosomal RNA and tRNA pseudouridine synthase RluA. J Biol Chem 274:18880–18886

    PubMed  Article  CAS  Google Scholar 

  39. Roeder GS, Keil RL, Voelkel-Meiman KA (1986) A recombination-stimulating sequence in the ribosomal RNA gene cluster of yeast. In: Klar A, Strathern JN (eds) Mechanisms of yeast recombination. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 29–33

    Google Scholar 

  40. Rong L, Palladino F, Aguilera A, Klein H (1991) The hyper-gene conversion hrp5-1 mutation of Saccharomyces cerevisiae is an allele of the SRS2/RADH gene. Genetics 127:75–85

    PubMed  CAS  Google Scholar 

  41. Rose MD, Novick P, Thomas JH, Botstein D, Fink GR (1987) A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene 60:237–243

    PubMed  Article  CAS  Google Scholar 

  42. Rose MD, Winston F, Hieter P (1990) Methods in Yeast genetics: a laboratory course manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  43. Ruggero D, Grisendi S, Piazza F, Rego E, Mari F, Rao PH, Cordon-Cardo C, Pandolfi PP (2003) Dyskeratosis congenita and cancer in mice deficient in ribosomal RNA modification. Science 299:259–262

    PubMed  Article  CAS  Google Scholar 

  44. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  45. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    PubMed  Article  CAS  Google Scholar 

  46. Schwindinger WF, Warner JR (1987) Transcriptional elements of the yeast ribosomal protein gene CYH2. J Biol Chem 262:5690–5695

    PubMed  CAS  Google Scholar 

  47. Simos G, Tekotte H, Grosjean H, Segref A, Sharma K, Tollervey D, Hurt EC (1996) Nuclear pore proteins are involved in the biogenesis of functional tRNA. EMBO J 15:2270–2284

    PubMed  CAS  Google Scholar 

  48. Stewart SE, Roeder GS (1989) Transcription by RNA polymerase I stimulates mitotic recombination in Saccharomyces cerevisiae. Mol Cell Biol 9:3464–3472

    PubMed  CAS  Google Scholar 

  49. Sung P, Trujillo KM, Van Komen S (2000) Recombination factors of Saccharomyces cerevisiae. Mutat Res 451:257–275

    PubMed  CAS  Google Scholar 

  50. Thompson DL, Kalderon D, Smith AE, Tevethia MJ (1990) Dissociation of Rb-binding and anchorage-independent growth from immortalization and tumorigenicity using SV40 mutants producing N-terminally truncated large T antigens. Virology 178:15–34

    PubMed  Article  CAS  Google Scholar 

  51. Voelkel-Meiman K, Roeder GS (1990) A chromosome containing HOT1 preferentially receives information during mitotic interchromosomal gene conversion. Genetics 124:561–572

    PubMed  CAS  Google Scholar 

  52. Voelkel-Meiman K, Keil RL, Roeder GS (1987) Recombination-stimulating sequences in yeast ribosomal DNA correspond to sequences regulating transcription by RNA polymerase I. Cell 48:1071–1079

    PubMed  Article  CAS  Google Scholar 

  53. Wai H, Johzuka K, Vu L, Eliason K, Kobayashi T, Horiuchi T, Nomura M (2001) Yeast RNA polymerase I enhancer is dispensable for transcription of the chromosomal rRNA gene and cell growth, and its apparent transcription enhancement from ectopic promoters requires Fob1 protein. Mol Cell Biol 21:5541–5553

    PubMed  Article  CAS  Google Scholar 

  54. Wolfe KH, Lohan AJ (1994) Sequence around the centromere of Saccharomyces cerevisiae chromosome II: similarity of CEN2 to CEN4. Yeast 10:S41–S46

    PubMed  Article  Google Scholar 

  55. Yarian C, Townsend H, Czestkowski W, Sochacka E, Malkiewicz AJ, Guenther R, Miskiewicz A, Agris PF (2002) Accurate translation of the genetic code depends on tRNA modified nucleotides. J Biol Chem 277:16391–16395

    PubMed  Article  CAS  Google Scholar 

  56. Yuan LW, Keil RL (1990) Distance-independence of mitotic intrachromosomal recombination in Saccharomyces cerevisiae. Genetics 124:263–273

    PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We thank Susan DiBartolomeis, Tom Reiner, Laura Palmer and Reeta Prusty for sharing their valuable technical expertise, and Anita Hopper for critical reading of the manuscript. Technical assistance from Jonathan Morgan and Paul Lewis is also appreciated. Plasmids containing deg1-D151A and controls were kindly provided by Henri Grosjean (CNRS, Laboratoire d‘Enzymologie et de Biochimie Structurales, France). This research was supported by funding from the Pennsylvania State System of Higher Education Faculty Professional Development Council and the Millersville University Faculty Grants Committee to CEH, a Sigma Xi Grant-in-Aid of Research to KGS, Millersville University Alumni Association Neimeyer-Hodgson Student Research Grants to MM, AR, GIL and DLH, and by National Institutes of Health grant GM-36422 to RLK.

Author information

Affiliations

Authors

Corresponding author

Correspondence to C. E. Hepfer.

Additional information

Communicated by A. Aguilera

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Hepfer, C.E., Arnold-Croop, S., Fogell, H. et al. DEG1, encoding the tRNA:pseudouridine synthase Pus3p, impacts HOT1-stimulated recombination in Saccharomyces cerevisiae . Mol Genet Genomics 274, 528–538 (2005). https://doi.org/10.1007/s00438-005-0042-3

Download citation

Keywords

  • HOT1
  • Recombination hotspot
  • DEG1
  • tRNA:pseudouridine synthase
  • Pus3p