Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1989 Jul;9(7):3081–3087. doi: 10.1128/mcb.9.7.3081

A single essential gene, PRI2, encodes the large subunit of DNA primase in Saccharomyces cerevisiae.

M Foiani 1, C Santocanale 1, P Plevani 1, G Lucchini 1
PMCID: PMC362777  PMID: 2528682

Abstract

DNA primase activity of the yeast DNA polymerase-primase complex is related to two polypeptides, p58 and p48. The reciprocal role of these protein species has not yet been clarified, although both participate in formation of the active center of the enzyme. The gene encoding the p58 subunit has been cloned by screening of a lambda gt11 yeast genomic DNA library, using specific anti-p58 antiserum. Antibodies that inhibited DNA primase activity could be purified by lysates of Escherichia coli cells infected with a recombinant bacteriophage containing the entire gene, which we designate PR12. The gene was found to be transcribed in a 1.7-kilobase mRNA whose level appeared to fluctuate during the mitotic cell cycle. Nucleotide sequence determination indicated that PR12 encodes a 528-amino-acid polypeptide with a calculated molecular weight of 62,262. The gene is unique in the haploid yeast genome, and its product is essential for cell viability, as has been shown for other components of the yeast DNA polymerase-primase complex.

Full text

PDF
3081

Images in this article

3082Image
on p.3082
3083Image
on p.3083
3083Image
on p.3083
3084Image
on p.3084
3085Image
on p.3085
3085Image
on p.3085

Selected References

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

  1. Barker D. G., White J. H., Johnston L. H. The nucleotide sequence of the DNA ligase gene (CDC9) from Saccharomyces cerevisiae: a gene which is cell-cycle regulated and induced in response to DNA damage. Nucleic Acids Res. 1985 Dec 9;13(23):8323–8337. doi: 10.1093/nar/13.23.8323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birkenmeyer L. G., Hill J. C., Dumas L. B. Saccharomyces cerevisiae CDC8 gene and its product. Mol Cell Biol. 1984 Apr;4(4):583–590. doi: 10.1128/mcb.4.4.583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Botstein D., Falco S. C., Stewart S. E., Brennan M., Scherer S., Stinchcomb D. T., Struhl K., Davis R. W. Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene. 1979 Dec;8(1):17–24. doi: 10.1016/0378-1119(79)90004-0. [DOI] [PubMed] [Google Scholar]
  4. Campbell J. L. Eukaryotic DNA replication. Annu Rev Biochem. 1986;55:733–771. doi: 10.1146/annurev.bi.55.070186.003505. [DOI] [PubMed] [Google Scholar]
  5. Carlson M., Botstein D. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell. 1982 Jan;28(1):145–154. doi: 10.1016/0092-8674(82)90384-1. [DOI] [PubMed] [Google Scholar]
  6. Foiani M., Lindner A. J., Hartmann G. R., Lucchini G., Plevani P. Affinity labeling of the active center and ribonucleoside triphosphate binding site of yeast DNA primase. J Biol Chem. 1989 Feb 5;264(4):2189–2194. [PubMed] [Google Scholar]
  7. Garnier J., Osguthorpe D. J., Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978 Mar 25;120(1):97–120. doi: 10.1016/0022-2836(78)90297-8. [DOI] [PubMed] [Google Scholar]
  8. Goto T., Wang J. C. Yeast DNA topoisomerase II is encoded by a single-copy, essential gene. Cell. 1984 Apr;36(4):1073–1080. doi: 10.1016/0092-8674(84)90057-6. [DOI] [PubMed] [Google Scholar]
  9. Hartwell L. H. Genetic control of the cell division cycle in yeast. II. Genes controlling DNA replication and its initiation. J Mol Biol. 1971 Jul 14;59(1):183–194. doi: 10.1016/0022-2836(71)90420-7. [DOI] [PubMed] [Google Scholar]
  10. Hattori M., Sakaki Y. Dideoxy sequencing method using denatured plasmid templates. Anal Biochem. 1986 Feb 1;152(2):232–238. doi: 10.1016/0003-2697(86)90403-3. [DOI] [PubMed] [Google Scholar]
  11. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  12. Hinnebusch A. G., Fink G. R. Repeated DNA sequences upstream from HIS1 also occur at several other co-regulated genes in Saccharomyces cerevisiae. J Biol Chem. 1983 Apr 25;258(8):5238–5247. [PubMed] [Google Scholar]
  13. Johnson L. M., Snyder M., Chang L. M., Davis R. W., Campbell J. L. Isolation of the gene encoding yeast DNA polymerase I. Cell. 1985 Nov;43(1):369–377. doi: 10.1016/0092-8674(85)90042-x. [DOI] [PubMed] [Google Scholar]
  14. Johnston L. H., White J. H., Johnson A. L., Lucchini G., Plevani P. 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. 1987 Jul 10;15(13):5017–5030. doi: 10.1093/nar/15.13.5017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kaguni L. S., Lehman I. R. Eukaryotic DNA polymerase-primase: structure, mechanism and function. Biochim Biophys Acta. 1988 Jul 13;950(2):87–101. doi: 10.1016/0167-4781(88)90001-2. [DOI] [PubMed] [Google Scholar]
  16. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Langford C. J., Klinz F. J., Donath C., Gallwitz D. Point mutations identify the conserved, intron-contained TACTAAC box as an essential splicing signal sequence in yeast. Cell. 1984 Mar;36(3):645–653. doi: 10.1016/0092-8674(84)90344-1. [DOI] [PubMed] [Google Scholar]
  19. Lucchini G., Brandazza A., Badaracco G., Bianchi M., Plevani P. Identification of the yeast DNA polymerase I gene with antibody probes. Curr Genet. 1985;10(4):245–252. doi: 10.1007/BF00365620. [DOI] [PubMed] [Google Scholar]
  20. Lucchini G., Francesconi S., Foiani M., Badaracco G., Plevani P. Yeast DNA polymerase--DNA primase complex; cloning of PRI 1, a single essential gene related to DNA primase activity. EMBO J. 1987 Mar;6(3):737–742. doi: 10.1002/j.1460-2075.1987.tb04815.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lucchini G., Mazza C., Scacheri E., Plevani P. Genetic mapping of the Saccharomyces cerevisiae DNA polymerase I gene and characterization of a pol1 temperature-sensitive mutant altered in DNA primase-polymerase complex stability. Mol Gen Genet. 1988 Jun;212(3):459–465. doi: 10.1007/BF00330850. [DOI] [PubMed] [Google Scholar]
  22. McIntosh E. M., Ord R. W., Storms R. K. Transcriptional regulation of the cell cycle-dependent thymidylate synthase gene of Saccharomyces cerevisiae. Mol Cell Biol. 1988 Nov;8(11):4616–4624. doi: 10.1128/mcb.8.11.4616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pabo C. O., Sauer R. T. Protein-DNA recognition. Annu Rev Biochem. 1984;53:293–321. doi: 10.1146/annurev.bi.53.070184.001453. [DOI] [PubMed] [Google Scholar]
  25. Pizzagalli A., Valsasnini P., Plevani P., Lucchini G. DNA polymerase I gene of Saccharomyces cerevisiae: nucleotide sequence, mapping of a temperature-sensitive mutation, and protein homology with other DNA polymerases. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3772–3776. doi: 10.1073/pnas.85.11.3772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Plevani P., Foiani M., Muzi Falconi M., Pizzagalli A., Santocanale C., Francesconi S., Valsasnini P., Comedini A., Piatti S., Lucchini G. The yeast DNA polymerase-primase complex: genes and proteins. Biochim Biophys Acta. 1988 Dec 20;951(2-3):268–273. doi: 10.1016/0167-4781(88)90096-6. [DOI] [PubMed] [Google Scholar]
  27. Plevani P., Foiani M., Valsasnini P., Badaracco G., Cheriathundam E., Chang L. M. Polypeptide structure of DNA primase from a yeast DNA polymerase-primase complex. J Biol Chem. 1985 Jun 10;260(11):7102–7107. [PubMed] [Google Scholar]
  28. Plevani P., Francesconi S., Lucchini G. The nucleotide sequence of the PRI1 gene related to DNA primase in Saccharomyces cerevisiae. Nucleic Acids Res. 1987 Oct 12;15(19):7975–7989. doi: 10.1093/nar/15.19.7975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Roeder G. S., Fink G. R. DNA rearrangements associated with a transposable element in yeast. Cell. 1980 Aug;21(1):239–249. doi: 10.1016/0092-8674(80)90131-2. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Scherer S., Davis R. W. Replacement of chromosome segments with altered DNA sequences constructed in vitro. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4951–4955. doi: 10.1073/pnas.76.10.4951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schwartz D. C., Cantor C. R. Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell. 1984 May;37(1):67–75. doi: 10.1016/0092-8674(84)90301-5. [DOI] [PubMed] [Google Scholar]
  33. Sharp P. M., Tuohy T. M., Mosurski K. R. Codon usage in yeast: cluster analysis clearly differentiates highly and lowly expressed genes. Nucleic Acids Res. 1986 Jul 11;14(13):5125–5143. doi: 10.1093/nar/14.13.5125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Shortle D., Haber J. E., Botstein D. Lethal disruption of the yeast actin gene by integrative DNA transformation. Science. 1982 Jul 23;217(4557):371–373. doi: 10.1126/science.7046050. [DOI] [PubMed] [Google Scholar]
  35. Smith D. E., Fisher P. A. Identification, developmental regulation, and response to heat shock of two antigenically related forms of a major nuclear envelope protein in Drosophila embryos: application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots. J Cell Biol. 1984 Jul;99(1 Pt 1):20–28. doi: 10.1083/jcb.99.1.20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Taylor G. R., Lagosky P. A., Storms R. K., Haynes R. H. Molecular characterization of the cell cycle-regulated thymidylate synthase gene of Saccharomyces cerevisiae. J Biol Chem. 1987 Apr 15;262(11):5298–5307. [PubMed] [Google Scholar]
  37. White J. H., Barker D. G., Nurse P., Johnston L. H. Periodic transcription as a means of regulating gene expression during the cell cycle: contrasting modes of expression of DNA ligase genes in budding and fission yeast. EMBO J. 1986 Jul;5(7):1705–1709. doi: 10.1002/j.1460-2075.1986.tb04414.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Winston F., Chumley F., Fink G. R. Eviction and transplacement of mutant genes in yeast. Methods Enzymol. 1983;101:211–228. doi: 10.1016/0076-6879(83)01016-2. [DOI] [PubMed] [Google Scholar]
  39. Wong S. W., Wahl A. F., Yuan P. M., Arai N., Pearson B. E., Arai K., Korn D., Hunkapiller M. W., Wang T. S. Human DNA polymerase alpha gene expression is cell proliferation dependent and its primary structure is similar to both prokaryotic and eukaryotic replicative DNA polymerases. EMBO J. 1988 Jan;7(1):37–47. doi: 10.1002/j.1460-2075.1988.tb02781.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Young R. A., Davis R. W. Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1194–1198. doi: 10.1073/pnas.80.5.1194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Young R. A., Davis R. W. Yeast RNA polymerase II genes: isolation with antibody probes. Science. 1983 Nov 18;222(4625):778–782. doi: 10.1126/science.6356359. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES