Skip to main content
SpringerLink
Log in

Suppressors of thermosensitive mutations in the DNA polymerase δ gene of Saccharomyces cerevisiae

  • Original Paper
  • Published:
Molecular and General Genetics MGG Aims and scope Submit manuscript

Abstract

DNA polymerases (Pol) α, δ and ε are necessary for replication of nuclear DNA. Po1δ interacts permanently or transiently with numerous accessory proteins whose identification may shed light on the function(s) of Po18. In vitro mutagenesis was used to induce thermosensitive (ts) mutations in the DNA polymerase δ gene (POL3). We have attempted to clone two recessive extragenic suppressors of such is mutants (sdp1 for mutation pol3-14 and sdp5-1 for mutation pol3-11) by transforming thermoresistant haploid strains pol3-14 sdpl and pol3-11 sdp5-1 with wild-type genomic libraries in singlecopy or multicopy vectors. None of the thermosensitive transformants so obtained was identified as being sdp1 or sdp5-1. Instead, three genes were cloned whose products interfere with the activity of suppressors. One of them is the type 1 protein phosphatase gene, D1S2. Another is a novel gene, ASM4, whose gene product is rich in asparagine and glutamine residues.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Bauer GA, Burgers PMJ (1990) Molecular cloning, structure and expression of the yeast proliferating cell nuclear antigen gene. Nucleic Acids Res 18:261–265

    Google Scholar 

  • Bauer GA, Heller HM, Burgers PMJ (1988) DNA polymerase III from Saccharomyces cerevisiae. Purification and characterization. J Biol Chem 263:917–924

    Google Scholar 

  • Boeke JD (1989) Transposable elements in Saccharomyces cerevisiae. In: Berg D, Howe M (eds) Mobile DNA. American Society for Microbiology, Washington, pp 335–374

    Google Scholar 

  • Boeke JD, Trueheart J, Natsoulis G, Fink GR (1987) 5-Fluoroorotic acid as a selection agent in yeast molecular genetics. Methods Enzymol 154:164–175

    Google Scholar 

  • Boulet A, Simon M, Faye G, Bauer G, Burgers PMJ (1989) Structure and function of the Saccharomyces cerevisiae CDC2 gene encoding the large subunit of DNA polymerase III. EMBO J 8:1849–1854

    Google Scholar 

  • Braithwaite D, Ito J (1993) Compilation, alignment and phylogenetic relationships of DNA polymerases. Nucleic Acids Res 21:787–802

    Google Scholar 

  • Broach J, Strathern JN, Hicks JB (1979) Transformation in yeast: development of hybrid cloning vector and isolation of the CAN1 gene. Gene 8:121–133

    Google Scholar 

  • Burgers PMJ (1991) Saccharomyces cerevisiae replication factor C. J Biol Chem 266:22698–22706

    Google Scholar 

  • Clotet J, Posas F, Casamayor A, Schaaff-Gerstenschlager I, Arino J (1991) The gene DIS2-S1 is essential in Saccharomyces cerevisiae and is involved in glycogen phosphorylase activation. Curr Genet 19:339–342

    Google Scholar 

  • Cullman G, Hindges R, Berchtold M, Hubscher U (1993) Cloning of mouse DNA polymerase 6 cDNA. Refinement of the homology boxes. Gene 139:191–200

    Google Scholar 

  • Decoster E, Vassal A, Faye G (1993) MSS1, a nuclear-encoded mitochondrial GTPase involved in the expression of COX1 subunit of cytochrome c oxidase. J Mol Biol 232:79–88

    Google Scholar 

  • Din SU, Brill SJ, Fairman MP, Stillman B (1990) Cell cycle-regulated phosphorylation of DNA replication factor-A from human and yeast cells. Genes Dev 4:968–977

    Google Scholar 

  • Doonan J, Morris NR (1989) The bimG gene of Aspergillus nidulans, required for completion of anaphase, encodes a homolog of mammalian phosphoprotein phosphatase 1. Cell 57:987–996

    Google Scholar 

  • Faye G, Fabre F, Simon M, Giot L, Boulet A, Bénit P, Vassal A (1992) DNA polymerase δ (POL3) of Saccharomyces cerevisiae. In: Hughes P, Fanning E, Kohiyama M (eds) DNA replication: the regulatory mechanisms. Springer Verlag, Berlin, pp 273–284

    Google Scholar 

  • 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

    Google Scholar 

  • Henikoff S (1987) Unidirectional digestion with exonuclease III in DNA sequence analysis. Methods Enzymol 155:156–165

    Google Scholar 

  • Johnston LH (1992) Cell cycle control of gene expression in yeast. Trends Cell Biol 2:353–357

    Google Scholar 

  • Kelman Z, O'Donnell M (1994) DNA replication: enzymology and mechanisms. Curr Opinion Genet Dev 4:185–195

    Google Scholar 

  • Linn S (1991) How many Pols does it take to replicate nuclear DNA? Cell 66:185–187

    Google Scholar 

  • Magni GE, von Borstel RC (1962) Different rates of spontaneous mutation during mitosis and meiosis in yeast. Genetics 47:1097–1108

    Google Scholar 

  • Matsuura A, Anraku Y (1994) Genetic interaction between the Ras-cAMP pathway and the Dis2s1/Glc7 protein phosphatase in Saccharomyces cerevisiae. Mol Gen Genet 242:257–262

    Google Scholar 

  • Morrison A, Sugino A (1993) DNA polymerase II, the epsilon polymerase of Saccharomyces cerevisiae. Prog Nucleic Acid Res Mol Biol 46:93–120

    Google Scholar 

  • Murray V (1989) Improved double-stranded DNA sequencing using the linear polymerase chain reaction. Nucleic Acids Res 17:88–89

    Google Scholar 

  • Nasheuer HP, Moore A, Wahl AF, Wang TSF (1991) Cell cycledependent phosphorylation of human DNA polymerase α. J Biol Chem 266:7893–7903

    Google Scholar 

  • Ohkura H, Kinoshita N, Miyatani S, Toda T, Yanagida M (1989) The fission yeast dis2+ gene required for chromosome disjoining encodes one of two putative type 1 protein phosphatases. Cell 57:997–1007

    Google Scholar 

  • Park H, Francesconi S, Wang TSF (1993) Cell cycle expression of two replicative DNA polymerases α and δ from Schizosaccharomyces pombe. Mol Biol Cell 4:145–157

    Google Scholar 

  • Rose MD, Fink GR (1987) KAR1, a gene required for function of both intranuclear and extranuclear microtubules in yeast. Cell 48:1047–1060

    Google Scholar 

  • Rose M, 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

    Google Scholar 

  • Rothstein R (1985) Cloning in yeast. In: Glover DM (ed) DNA cloning, a practical approach, volume II. IRL Press, Oxford, pp 45–65

    Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Schiestl RH, Gietz RD (1989) High efficiency transformation of intact yeast cells using single-stranded nucleic acids as a carrier. Curr Genet 16:339–346

    Google Scholar 

  • Sharp PM, Cowe E (1991) Synonymous codon usage in Saccharomyces cerevisiae. Yeast 7:657–678

    Google Scholar 

  • Sherman F (1991) Getting started with yeast. Methods Enzymol 194:3–21

    Google Scholar 

  • Sherman F, Fink G, Hicks J (1983) Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Sikorski RS, Boeke JD (1991) In vitro mutagenesis and plasmid shuffling from cloned gene to mutant yeast. Methods Enzymol 194:302–318

    Google Scholar 

  • Simon M, Giot L, Faye G (1991) The 3′ to 5′ exonuclease activity located in the DNA polymerase δ subunit of Saccharomyces cerevisiae is required for accurate replication. EMBO J 10:2165–2170

    Google Scholar 

  • Simon M, Giot L, Faye G (1993) A random mutagenesis procedure: application to the POL3 gene of Saccharomyces cerevisiae. Gene 127:139–144

    Google Scholar 

  • Simon M, Bénit P, Vassal A, Dubois C, Faye G (1994) Sequence of the PHO2-POL3(CDC2) region of chromosome IV of Saccharomyces cerevisiae. Yeast (in press)

  • Singh (1977) Nonsense suppressors of yeast cause osmotic-sensitive growth. Proc Natl Acad Sci USA 74:305–309

    Google Scholar 

  • Stettler S, Chiannilkulchai N, Hermann, Le Denmat S, Lalo D, Lacroute F, Sentenac A, Thuriaux P (1993) A general suppressor of RNA polymerase I, II and III mutations in Saccharomyces cerevisiae. Mol Gen Genet 239:169–176

    Google Scholar 

  • St John TP, Scherer S, McDonell M, Davis RW (1981) Deletion analysis of the Saccharomyces Gal gene cluster. Transcription from three promoters. J Mol Biol 152:317–334

    Google Scholar 

  • Struhl K (1985) Nucleotide sequence and transcriptional mapping of the yeast pet56-his3-dedl gene region. Nucleic Acids Res 13:8587–8601

    Google Scholar 

  • Struhl K, Stinchcomb DT, Scherer S, Davis RW (1979) High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci USA 76:1471–1475

    Google Scholar 

  • Thomas JH, Botstein D (1986) A gene required for the separation of chromosomes on the spindle apparatus in yeast. Cell 44:65–76

    Google Scholar 

  • Tschumper G, Carbon J (1980) Sequence of a yeast DNA fragment containing a chromosomal replicator and the TRP1 gene. Gene 10:157–166

    Google Scholar 

  • Waga S, Stillman B (1994) Anatomy of a DNA replication fork revealed by reconstitution of SV40 DNA replication in vitro. Nature 369:207–212

    Google Scholar 

  • Wang TSF (1991) Eukaryotic DNA polymerases. Annu Rev Biochem 60:513–552

    Google Scholar 

  • Yoshimoto H, Yamashita I (1991) The GAM1/SNF2 gene of Saccharomyces cerevisiae encodes a highly charged nuclear protein required for transcription of the STA1 gene. Mol Gen Genet 228:270–280

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institut Curie-Biologie, Centre Universitaire, Bât. 110, 91405, Orsay Cedex, France

    Loic Giot, Michel Simon, Celine Dubois & Gerard Faye

Authors
  1. Loic Giot
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michel Simon
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Celine Dubois
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gerard Faye
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Communicated by B. J. Kilbey

Rights and permissions

Reprints and permissions

About this article

Cite this article

Giot, L., Simon, M., Dubois, C. et al. Suppressors of thermosensitive mutations in the DNA polymerase δ gene of Saccharomyces cerevisiae . Molec. Gen. Genet. 246, 212–222 (1995). https://doi.org/10.1007/BF00294684

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00294684

Key words

Search

Navigation