Skip to main content
Log in

Genetic mapping of the Saccharomyces cerevisiae DNA polymerase I gene and characterization of a pol1 temperaturesensitive mutant altered in DNA primase-polymerase complex stability

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

Summary

The cloned DNA polymerase I gene has been used to map the POL1 locus on the left arm of chromosome XIV, between MET4 and TOP2. Temperature-sensitive mutants in POL1 have been obtained by in vitro mutagenesis of the cloned gene and in vivo replacement of the wild-type allele with the mutated copy. Physiological and biochemical characterization of one temperature-sensitive mutant (pol1-1) shows that cells shifted to the non-permissive temperature can complete one round of cell division and DNA replication before they arrest. Analysis of DNA polymerase I in crude extracts and in partially purified preparations indicates that the pol1-1 mutation results in a conformational change and affects the stability of the DNA primase-polymerase complex.

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

Access this article

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

Instant access to the full article PDF.

Similar content being viewed by others

References

  • Badaracco G, Capucci L, Plevani P, Chang LMS (1983) Polypeptide structure of DNA polymerase I from Saccharomyces cerevisiae. J Biol Chem 258:10720–10726

    Google Scholar 

  • Bisson L, Thorner J (1977) Thymidine 5′-monophosphate-requiring mutants of Saccharomyces cerevisiae are deficient in thymidylate synthetase. J Bacteriol 132:44–50

    Google Scholar 

  • Boeke JD, La Croute F, Fink GR (1984) A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197:345–346

    Google Scholar 

  • Botstein D, Falco SC, Steward SE, Brennan M, Scherer S, Stinchcomb DT, Struhl K, Davis RW (1979) Sterile host yeast (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene 8:17–24

    Google Scholar 

  • Budd M, Campbell JL (1987) Temperature-sensitive mutations in the yeast DNA polymerase I gene. Proc Natl Acad Sci USA 84:2838–2842

    Google Scholar 

  • Campbell JL (1986) Eukaryotic DNA replication. Ann Rev Biochem 55:733–771

    Google Scholar 

  • Chang LMS (1977) DNA polymerase from bakers yeast. J Biol Chem 252:1873–1880

    Google Scholar 

  • Culotti J, Hartwell LH (1971) Genetic control of the cell division cycle in yeast. Exp Cell Res 67:389–401

    Google Scholar 

  • Donahue TF, Daves RS, Lucchini G, Fink GR (1983) A short nucleotide sequence required for regulation of HIS4 by the general control system in yeast. Cell 32:89–98

    Google Scholar 

  • Dumas LB, Lussky JP, Mc Farland EJ, Shampay J (1982) New temperature-sensitive mutants of S. cerevisiae affecting DNA replication. Mol Gen Genet 187:42–46

    Google Scholar 

  • Game J (1976) Yeast cell cycle mutant cdc21 is a temperaturesensitive thymidylate auxotroph. Mol Gen Genet 146:313–315

    Google Scholar 

  • Hartwell LH (1971) Genetic control of the cell division cycle in yeast. J Mol Biol 59:183–194

    Google Scholar 

  • Hartwell L (1976) Sequential functions of gene products relative to DNA synthesis in the yeast cell cycle. J Mol Biol 104:803–817

    Google Scholar 

  • Hinnen A, Hicks JA, Fink GR (1978) Transformation of yeast. Proc Natl Acad Sci USA 75:1929–1933

    Google Scholar 

  • Holm C, Goto T, Wang JC, Botstein D (1985) DNA topoisomerase II is required at the time of mitosis in yeast. Cell 41:553–563

    Google Scholar 

  • Johnson LM, Snyder M, Chang LMS, Davis RW, Campbell JL (1985) Identification of the gene encoding yeast DNA polymerase I. Cell 43:369–377

    Google Scholar 

  • Johnston LH, Game JC (1978) Mutants in yeast with depressed DNA synthesis. Mol Gen Genet 161:205–214

    Google Scholar 

  • Johnston LH, Nasmyth KA (1978) S. cerevisiae cell cycle mutant cdc9 is defective in DNA ligase. Nature 274:891–894

    Google Scholar 

  • Johnston LH, White JHM, Johnson AL, Lucchini G, Plevani P (1987) The yeast DNA polymerase I transcript is regulated in both the mitotic cell cycle and in meiosis and is also induced after DNA damage. Nucleic Acids Res 15:5017–5029

    Google Scholar 

  • Jong AYS, Kuo CL, Campbell JL (1984) The CDC8 gene of yeast encode thymidylate kinase. J Biol Chem 259:11052–11059

    Google Scholar 

  • Kirshbaum JB, Claeys IV, Nasi S, Molholt B, Miller JH (1975) Temperature-sensitive RNA polymerase mutants with altered subunit synthesis and degradation. Proc Natl Acad Sci USA 72:2375–2379

    Google Scholar 

  • Kuo CL, Huang I, Campbell JL (1983) Isolation of yeast DNA replication mutants in permeabilized cells. Proc Natl Acad Sci USA 80:6465–6469

    Google Scholar 

  • Lucchini G, Brandazza A, Badaracco G, Bianchi M, Plevani P (1985) Identification of the yeast DNA polymerase I gene with antibody probes. Curr Genet 10:245–252

    Google Scholar 

  • Mortimer RK, Hawthorne DC (1969) Yeast genetics. In: Rose AH, Harryson JS (eds) The yeast, vol 1. Academic Press, New York, pp 385–460

    Google Scholar 

  • Orr-Weaver TL, Szostak JW, Rothstein RJ (1981) Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci USA 78:6354–6358

    Google Scholar 

  • Plevani P, Badaracco G, Augl C, Chang LMS (1984) DNA polymerase I and DNA primase complex in yeast. J Biol Chem 259:7532–7539

    Google Scholar 

  • Plevani P, Foiani M, Valsasnini P, Badaracco G, Cheriathundam E, Chang LMS (1985) Polypeptide structure of DNA primase from a yeast DNA polymerase: primase complex. J Biol Chem 260:7102–7107

    Google Scholar 

  • Plevani P, Lucchini G, Foiani M, Valsasnini P, Brandazza A, Bianchi M, Magni G, Badaracco G (1987) Structure and function of the yeast DNA polymerase I-DNA primase complex. Mol Genet (Life Sci Adv) 6:53–60

    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 

  • Schwartz DC, Cantor CR (1984) Separation of yeast chromosomesized DNAs by pulsed field gradient gel electrophoresis. Cell 37:67–75

    Google Scholar 

  • Sclafani RA, Fangman W (1984) Yeast gene CDC8 encodes thymidylate kinase and is complemented by herpes thymidine kinase gene TK. Proc Natl Acad Sci USA 81:5821–5825

    Google Scholar 

  • Sherman F, Fink GR, Hicks JH (1986) Methods in yeast genetics. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Shortle D, Novick P, Botstein D (1984) Construction and genetic characterization of temperature-sensitive mutant alleles of the yeast actin gene. Proc Natl Acad Sci USA 81:4889–4893

    Google Scholar 

  • Singh H, Dumas LB (1984) A DNA primase that copurifies with the major DNA polymerase from the yeast Saccharomyces cerevisiae. J Biol Chem 259:7936–7940

    Google Scholar 

  • Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517

    Google Scholar 

  • Taketo M, Ishihama A (1976) Biosynthesis of RNA polymerase in Escherichia coli IV. Accumulation of intermediates in mutants defective in subunit assembly. J Mol Biol 102:297–310

    Google Scholar 

  • Wintersberger U, Wintersberger E (1970) Studies of deoxyribonucleic acid polymerases from yeast. Eur J Biochem 13:11–19

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Additional information

Communicated by G. Fink

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lucchini, G., Mazza, C., Scacheri, E. et al. Genetic mapping of the Saccharomyces cerevisiae DNA polymerase I gene and characterization of a pol1 temperaturesensitive mutant altered in DNA primase-polymerase complex stability. Mol Gen Genet 212, 459–465 (1988). https://doi.org/10.1007/BF00330850

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00330850

Key words

Navigation