Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1987 Mar;6(3):737–742. doi: 10.1002/j.1460-2075.1987.tb04815.x

Yeast DNA polymerase--DNA primase complex; cloning of PRI 1, a single essential gene related to DNA primase activity.

G Lucchini, S Francesconi, M Foiani, G Badaracco, P Plevani
PMCID: PMC553458  PMID: 3034609

Abstract

The immunopurified yeast DNA polymerase--DNA primase complex is constituted by DNA polymerase I polypeptides and by three other protein species, called p74, p58 and p48, which we show to be immunologically unrelated. The gene encoding the p48 polypeptide has been identified by immunological screening of a lambda gt11 yeast genomic DNA library. Antiserum specific for p48 inhibits DNA primase, and immunoreactive, inhibitory antibodies are affinity-purified by the clone-encoded protein, thus relating the p48 polypeptide to DNA primase activity. The entire gene has been cloned, and the 1.45-kb p48 mRNA is overproduced in cells containing the gene in high copy number. Gene disruption and Southern hybridization experiments demonstrate that the p48 protein is encoded by a single gene and it performs an essential function.

Full text

PDF
737

Images in this article

738Fig. 1.
on p.738
738Fig. 2.
on p.738
739Fig. 3.
on p.739
739Fig. 4.
on p.739
739Fig. 5.
on p.739
740Fig. 7.
on p.740

Selected References

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

  1. Badaracco G., Bianchi M., Valsasnini P., Magni G., Plevani P. Initiation, elongation and pausing of in vitro DNA synthesis catalyzed by immunopurified yeast DNA primase: DNA polymerase complex. EMBO J. 1985 May;4(5):1313–1317. doi: 10.1002/j.1460-2075.1985.tb03778.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Carle G. F., Olson M. V. An electrophoretic karyotype for yeast. Proc Natl Acad Sci U S A. 1985 Jun;82(11):3756–3760. doi: 10.1073/pnas.82.11.3756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Chang L. M. DNA polymerases from bakers' yeast. J Biol Chem. 1977 Mar 25;252(6):1873–1880. [PubMed] [Google Scholar]
  6. Chang L. M., Rafter E., Augl C., Bollum F. J. Purification of a DNA polymerase-DNA primase complex from calf thymus glands. J Biol Chem. 1984 Dec 10;259(23):14679–14687. [PubMed] [Google Scholar]
  7. Conaway R. C., Lehman I. R. A DNA primase activity associated with DNA polymerase alpha from Drosophila melanogaster embryos. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2523–2527. doi: 10.1073/pnas.79.8.2523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. 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]
  11. Kaguni L. S., Rossignol J. M., Conaway R. C., Lehman I. R. Isolation of an intact DNA polymerase-primase from embryos of Drosophila melanogaster. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2221–2225. doi: 10.1073/pnas.80.8.2221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. 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]
  15. Plevani P., Badaracco G., Augl C., Chang L. M. DNA polymerase I and DNA primase complex in yeast. J Biol Chem. 1984 Jun 25;259(12):7532–7539. [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. 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]
  19. Shioda M., Nelson E. M., Bayne M. L., Benbow R. M. DNA primase activity associated with DNA polymerase alpha from Xenopus laevis ovaries. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7209–7213. doi: 10.1073/pnas.79.23.7209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Singh H., Dumas L. B. A DNA primase that copurifies with the major DNA polymerase from the yeast Saccharomyces cerevisiae. J Biol Chem. 1984 Jun 25;259(12):7936–7940. [PubMed] [Google Scholar]
  22. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  23. Tseng B. Y., Ahlem C. N. A DNA primase from mouse cells. Purification and partial characterization. J Biol Chem. 1983 Aug 25;258(16):9845–9849. [PubMed] [Google Scholar]
  24. Tseng B. Y., Ahlem C. N. Mouse primase initiation sites in the origin region of simian virus 40. Proc Natl Acad Sci U S A. 1984 Apr;81(8):2342–2346. doi: 10.1073/pnas.81.8.2342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Vaitukaitis J. L. Production of antisera with small doses of immunogen: multiple intradermal injections. Methods Enzymol. 1981;73(Pt B):46–52. doi: 10.1016/0076-6879(81)73055-6. [DOI] [PubMed] [Google Scholar]
  26. Wang T. S., Hu S. Z., Korn D. DNA primase from KB cells. Characterization of a primase activity tightly associated with immunoaffinity-purified DNA polymerase-alpha. J Biol Chem. 1984 Feb 10;259(3):1854–1865. [PubMed] [Google Scholar]
  27. 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]
  28. Wintersberger U., Wintersberger E. Studies on deoxyribonucleic acid polymerases from yeast. 1. Parial purification and properties of two DNA polymerases from mitochondria-free cell extracts. Eur J Biochem. 1970 Mar 1;13(1):11–19. doi: 10.1111/j.1432-1033.1970.tb00893.x. [DOI] [PubMed] [Google Scholar]
  29. Yagura T., Kozu T., Seno T. Mouse DNA replicase. DNA polymerase associated with a novel RNA polymerase activity to synthesize initiator RNA of strict size. J Biol Chem. 1982 Sep 25;257(18):11121–11127. [PubMed] [Google Scholar]
  30. 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]
  31. 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 The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES