Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1995 Jul 3;14(13):3094–3104. doi: 10.1002/j.1460-2075.1995.tb07312.x

hsk1+, a Schizosaccharomyces pombe gene related to Saccharomyces cerevisiae CDC7, is required for chromosomal replication.

H Masai 1, T Miyake 1, K Arai 1
PMCID: PMC394371  PMID: 7621824

Abstract

Degenerate oligonucleotide-directed polymerase chain reaction was conducted to clone a possible Schizosaccharomyces pombe homologue [hsk1 for a putative homologue of CDC7 (seven) kinase 1] of Saccharomyces cerevisiae Cdc7 kinase. The cloned cDNA for hsk1+ contains an open reading frame consisting of 507 amino acids with predicted mol. wt of 58,370 that possesses overall amino acid identity of 46% (65% including similar residues) to CDC7. In addition to conserved domains for serine-threonine kinases, the predicted primary structure of Hsk1 contains three 'kinase insert' sequences characteristic to Cdc7 at the positions identical to those of Cdc7. Whereas the length and sequences of the kinase inserts are diverged between the two yeast species, 58% identity (76% including similar residues) is detected within the kinase conserved domains. The hsk1+ gene, which is present as a single copy on the S.pombe chromosome, contains two introns within the coding frame. Disruption of the hsk1+ gene by insertion of the ura4+ gene is lethal to growth. Analysis of the DNA content of germinating spores that contain hsk1 null alleles indicates that DNA replication is inhibited in the mutant. The morphology of these mutant spores after germination indicates abnormal nuclear division in some population of germinating spores, suggesting either that Hsk1 may be required for inhibition of mitosis until completion of S phase or that it may also be involved in proper execution of mitosis. Our results suggest that hsk1+ is a strong candidate for the functional fission yeast homologue of budding yeast CDC7 and that a mechanism through which initiation of chromosomal replication is regulated may be conserved between the two yeast species.

Full text

PDF
3099

Images in this article

3096Image
on p.3096
3097Image
on p.3097
3097Image
on p.3097
3099Image
on p.3099

Selected References

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

  1. Andrews B. J., Herskowitz I. Regulation of cell cycle-dependent gene expression in yeast. J Biol Chem. 1990 Aug 25;265(24):14057–14060. [PubMed] [Google Scholar]
  2. Bell S. P., Kobayashi R., Stillman B. Yeast origin recognition complex functions in transcription silencing and DNA replication. Science. 1993 Dec 17;262(5141):1844–1849. doi: 10.1126/science.8266072. [DOI] [PubMed] [Google Scholar]
  3. Bell S. P., Stillman B. ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex. Nature. 1992 May 14;357(6374):128–134. doi: 10.1038/357128a0. [DOI] [PubMed] [Google Scholar]
  4. Brenner C., Fuller R. S. Structural and enzymatic characterization of a purified prohormone-processing enzyme: secreted, soluble Kex2 protease. Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):922–926. doi: 10.1073/pnas.89.3.922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Caddle M. S., Calos M. P. Specific initiation at an origin of replication from Schizosaccharomyces pombe. Mol Cell Biol. 1994 Mar;14(3):1796–1805. doi: 10.1128/mcb.14.3.1796. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DePamphilis M. L. Eukaryotic DNA replication: anatomy of an origin. Annu Rev Biochem. 1993;62:29–63. doi: 10.1146/annurev.bi.62.070193.000333. [DOI] [PubMed] [Google Scholar]
  7. Dowell S. J., Romanowski P., Diffley J. F. Interaction of Dbf4, the Cdc7 protein kinase regulatory subunit, with yeast replication origins in vivo. Science. 1994 Aug 26;265(5176):1243–1246. doi: 10.1126/science.8066465. [DOI] [PubMed] [Google Scholar]
  8. Dubey D. D., Zhu J., Carlson D. L., Sharma K., Huberman J. A. Three ARS elements contribute to the ura4 replication origin region in the fission yeast, Schizosaccharomyces pombe. EMBO J. 1994 Aug 1;13(15):3638–3647. doi: 10.1002/j.1460-2075.1994.tb06671.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fikes J. D., Becker D. M., Winston F., Guarente L. Striking conservation of TFIID in Schizosaccharomyces pombe and Saccharomyces cerevisiae. Nature. 1990 Jul 19;346(6281):291–294. doi: 10.1038/346291a0. [DOI] [PubMed] [Google Scholar]
  10. Foss M., McNally F. J., Laurenson P., Rine J. Origin recognition complex (ORC) in transcriptional silencing and DNA replication in S. cerevisiae. Science. 1993 Dec 17;262(5141):1838–1844. doi: 10.1126/science.8266071. [DOI] [PubMed] [Google Scholar]
  11. Francesconi S., Park H., Wang T. S. Fission yeast with DNA polymerase delta temperature-sensitive alleles exhibits cell division cycle phenotype. Nucleic Acids Res. 1993 Aug 11;21(16):3821–3828. doi: 10.1093/nar/21.16.3821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gietz D., St Jean A., Woods R. A., Schiestl R. H. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 1992 Mar 25;20(6):1425–1425. doi: 10.1093/nar/20.6.1425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hanks S. K., Quinn A. M., Hunter T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science. 1988 Jul 1;241(4861):42–52. doi: 10.1126/science.3291115. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Hartwell L. H. Saccharomyces cerevisiae cell cycle. Bacteriol Rev. 1974 Jun;38(2):164–198. doi: 10.1128/br.38.2.164-198.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hartwell L. H. Three additional genes required for deoxyribonucleic acid synthesis in Saccharomyces cerevisiae. J Bacteriol. 1973 Sep;115(3):966–974. doi: 10.1128/jb.115.3.966-974.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hartwell L. H., Weinert T. A. Checkpoints: controls that ensure the order of cell cycle events. Science. 1989 Nov 3;246(4930):629–634. doi: 10.1126/science.2683079. [DOI] [PubMed] [Google Scholar]
  18. Hindley J., Phear G. A. Sequence of the cell division gene CDC2 from Schizosaccharomyces pombe; patterns of splicing and homology to protein kinases. Gene. 1984 Nov;31(1-3):129–134. doi: 10.1016/0378-1119(84)90203-8. [DOI] [PubMed] [Google Scholar]
  19. Hirano T., Funahashi S., Uemura T., Yanagida M. Isolation and characterization of Schizosaccharomyces pombe cutmutants that block nuclear division but not cytokinesis. EMBO J. 1986 Nov;5(11):2973–2979. doi: 10.1002/j.1460-2075.1986.tb04594.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hofmann J. F., Beach D. cdt1 is an essential target of the Cdc10/Sct1 transcription factor: requirement for DNA replication and inhibition of mitosis. EMBO J. 1994 Jan 15;13(2):425–434. doi: 10.1002/j.1460-2075.1994.tb06277.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hollingsworth R. E., Jr, Ostroff R. M., Klein M. B., Niswander L. A., Sclafani R. A. Molecular genetic studies of the Cdc7 protein kinase and induced mutagenesis in yeast. Genetics. 1992 Sep;132(1):53–62. doi: 10.1093/genetics/132.1.53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hollingsworth R. E., Jr, Sclafani R. A. DNA metabolism gene CDC7 from yeast encodes a serine (threonine) protein kinase. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6272–6276. doi: 10.1073/pnas.87.16.6272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hood M. T., Stachow C. Transformation of Schizosaccharomyces pombe by electroporation. Nucleic Acids Res. 1990 Feb 11;18(3):688–688. doi: 10.1093/nar/18.3.688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Huberman J. A., Zhu J. G., Davis L. R., Newlon C. S. Close association of a DNA replication origin and an ARS element on chromosome III of the yeast, Saccharomyces cerevisiae. Nucleic Acids Res. 1988 Jul 25;16(14A):6373–6384. doi: 10.1093/nar/16.14.6373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Inoue H., Nojima H., Okayama H. High efficiency transformation of Escherichia coli with plasmids. Gene. 1990 Nov 30;96(1):23–28. doi: 10.1016/0378-1119(90)90336-p. [DOI] [PubMed] [Google Scholar]
  26. Jackson A. L., Pahl P. M., Harrison K., Rosamond J., Sclafani R. A. Cell cycle regulation of the yeast Cdc7 protein kinase by association with the Dbf4 protein. Mol Cell Biol. 1993 May;13(5):2899–2908. doi: 10.1128/mcb.13.5.2899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Jazwinski S. M. CDC7-dependent protein kinase activity in yeast replicative-complex preparations. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2101–2105. doi: 10.1073/pnas.85.7.2101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Johnston L. H., Thomas A. P. A further two mutants defective in initiation of the S phase in the yeast Saccharomyces cerevisiae. Mol Gen Genet. 1982;186(3):445–448. doi: 10.1007/BF00729467. [DOI] [PubMed] [Google Scholar]
  29. Johnston L. H., Thomas A. P. The isolation of new DNA synthesis mutants in the yeast Saccharomyces cerevisiae. Mol Gen Genet. 1982;186(3):439–444. doi: 10.1007/BF00729466. [DOI] [PubMed] [Google Scholar]
  30. Kelly T. J., Martin G. S., Forsburg S. L., Stephen R. J., Russo A., Nurse P. The fission yeast cdc18+ gene product couples S phase to START and mitosis. Cell. 1993 Jul 30;74(2):371–382. doi: 10.1016/0092-8674(93)90427-r. [DOI] [PubMed] [Google Scholar]
  31. Kitada K., Johnston L. H., Sugino T., Sugino A. Temperature-sensitive cdc7 mutations of Saccharomyces cerevisiae are suppressed by the DBF4 gene, which is required for the G1/S cell cycle transition. Genetics. 1992 May;131(1):21–29. doi: 10.1093/genetics/131.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kleinschmidt J. A., Dingwall C., Maier G., Franke W. W. Molecular characterization of a karyophilic, histone-binding protein: cDNA cloning, amino acid sequence and expression of nuclear protein N1/N2 of Xenopus laevis. EMBO J. 1986 Dec 20;5(13):3547–3552. doi: 10.1002/j.1460-2075.1986.tb04681.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kozak M. The scanning model for translation: an update. J Cell Biol. 1989 Feb;108(2):229–241. doi: 10.1083/jcb.108.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Krebs E. G. The growth of research on protein phosphorylation. Trends Biochem Sci. 1994 Nov;19(11):439–439. doi: 10.1016/0968-0004(94)90125-2. [DOI] [PubMed] [Google Scholar]
  35. Leonhardt H., Page A. W., Weier H. U., Bestor T. H. A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei. Cell. 1992 Nov 27;71(5):865–873. doi: 10.1016/0092-8674(92)90561-p. [DOI] [PubMed] [Google Scholar]
  36. Li J. J., Herskowitz I. Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system. Science. 1993 Dec 17;262(5141):1870–1874. doi: 10.1126/science.8266075. [DOI] [PubMed] [Google Scholar]
  37. Lowndes N. F., McInerny C. J., Johnson A. L., Fantes P. A., Johnston L. H. Control of DNA synthesis genes in fission yeast by the cell-cycle gene cdc10+. Nature. 1992 Jan 30;355(6359):449–453. doi: 10.1038/355449a0. [DOI] [PubMed] [Google Scholar]
  38. Løbner-Olesen A., Skarstad K., Hansen F. G., von Meyenburg K., Boye E. The DnaA protein determines the initiation mass of Escherichia coli K-12. Cell. 1989 Jun 2;57(5):881–889. doi: 10.1016/0092-8674(89)90802-7. [DOI] [PubMed] [Google Scholar]
  39. Marahrens Y., Stillman B. A yeast chromosomal origin of DNA replication defined by multiple functional elements. Science. 1992 Feb 14;255(5046):817–823. doi: 10.1126/science.1536007. [DOI] [PubMed] [Google Scholar]
  40. Marchuk D., Drumm M., Saulino A., Collins F. S. Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res. 1991 Mar 11;19(5):1154–1154. doi: 10.1093/nar/19.5.1154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Maundrell K., Hutchison A., Shall S. Sequence analysis of ARS elements in fission yeast. EMBO J. 1988 Jul;7(7):2203–2209. doi: 10.1002/j.1460-2075.1988.tb03059.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Maundrell K. Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene. 1993 Jan 15;123(1):127–130. doi: 10.1016/0378-1119(93)90551-d. [DOI] [PubMed] [Google Scholar]
  43. Maundrell K. nmt1 of fission yeast. A highly transcribed gene completely repressed by thiamine. J Biol Chem. 1990 Jul 5;265(19):10857–10864. [PubMed] [Google Scholar]
  44. McIntosh E. M., Atkinson T., Storms R. K., Smith M. Characterization of a short, cis-acting DNA sequence which conveys cell cycle stage-dependent transcription in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Jan;11(1):329–337. doi: 10.1128/mcb.11.1.329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Mertins P., Gallwitz D. Nuclear pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe strictly requires an intron-contained, conserved sequence element. EMBO J. 1987 Jun;6(6):1757–1763. doi: 10.1002/j.1460-2075.1987.tb02428.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Micklem G., Rowley A., Harwood J., Nasmyth K., Diffley J. F. Yeast origin recognition complex is involved in DNA replication and transcriptional silencing. Nature. 1993 Nov 4;366(6450):87–89. doi: 10.1038/366087a0. [DOI] [PubMed] [Google Scholar]
  47. Miyajima I., Nakafuku M., Nakayama N., Brenner C., Miyajima A., Kaibuchi K., Arai K., Kaziro Y., Matsumoto K. GPA1, a haploid-specific essential gene, encodes a yeast homolog of mammalian G protein which may be involved in mating factor signal transduction. Cell. 1987 Sep 25;50(7):1011–1019. doi: 10.1016/0092-8674(87)90167-x. [DOI] [PubMed] [Google Scholar]
  48. Moreno S., Klar A., Nurse P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 1991;194:795–823. doi: 10.1016/0076-6879(91)94059-l. [DOI] [PubMed] [Google Scholar]
  49. Newlon C. S., Theis J. F. The structure and function of yeast ARS elements. Curr Opin Genet Dev. 1993 Oct;3(5):752–758. doi: 10.1016/s0959-437x(05)80094-2. [DOI] [PubMed] [Google Scholar]
  50. Nishizuka Y. The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature. 1988 Aug 25;334(6184):661–665. doi: 10.1038/334661a0. [DOI] [PubMed] [Google Scholar]
  51. Nurse P. Ordering S phase and M phase in the cell cycle. Cell. 1994 Nov 18;79(4):547–550. doi: 10.1016/0092-8674(94)90539-8. [DOI] [PubMed] [Google Scholar]
  52. Okazaki K., Okazaki N., Kume K., Jinno S., Tanaka K., Okayama H. High-frequency transformation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of Schizosaccharomyces pombe. Nucleic Acids Res. 1990 Nov 25;18(22):6485–6489. doi: 10.1093/nar/18.22.6485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Patterson M., Sclafani R. A., Fangman W. L., Rosamond J. Molecular characterization of cell cycle gene CDC7 from Saccharomyces cerevisiae. Mol Cell Biol. 1986 May;6(5):1590–1598. doi: 10.1128/mcb.6.5.1590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Rao H., Marahrens Y., Stillman B. Functional conservation of multiple elements in yeast chromosomal replicators. Mol Cell Biol. 1994 Nov;14(11):7643–7651. doi: 10.1128/mcb.14.11.7643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Rivier D. H., Rine J. An origin of DNA replication and a transcription silencer require a common element. Science. 1992 May 1;256(5057):659–663. doi: 10.1126/science.1585179. [DOI] [PubMed] [Google Scholar]
  56. Russell D. W., Zinder N. D. Hemimethylation prevents DNA replication in E. coli. Cell. 1987 Sep 25;50(7):1071–1079. doi: 10.1016/0092-8674(87)90173-5. [DOI] [PubMed] [Google Scholar]
  57. Saka Y., Fantes P., Sutani T., McInerny C., Creanor J., Yanagida M. Fission yeast cut5 links nuclear chromatin and M phase regulator in the replication checkpoint control. EMBO J. 1994 Nov 15;13(22):5319–5329. doi: 10.1002/j.1460-2075.1994.tb06866.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Saka Y., Yanagida M. Fission yeast cut5+, required for S phase onset and M phase restraint, is identical to the radiation-damage repair gene rad4+. Cell. 1993 Jul 30;74(2):383–393. doi: 10.1016/0092-8674(93)90428-s. [DOI] [PubMed] [Google Scholar]
  59. Sclafani R. A., Patterson M., Rosamond J., Fangman W. L. Differential regulation of the yeast CDC7 gene during mitosis and meiosis. Mol Cell Biol. 1988 Jan;8(1):293–300. doi: 10.1128/mcb.8.1.293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Sekimizu K., Bramhill D., Kornberg A. ATP activates dnaA protein in initiating replication of plasmids bearing the origin of the E. coli chromosome. Cell. 1987 Jul 17;50(2):259–265. doi: 10.1016/0092-8674(87)90221-2. [DOI] [PubMed] [Google Scholar]
  61. Shirahige K., Iwasaki T., Rashid M. B., Ogasawara N., Yoshikawa H. Location and characterization of autonomously replicating sequences from chromosome VI of Saccharomyces cerevisiae. Mol Cell Biol. 1993 Aug;13(8):5043–5056. doi: 10.1128/mcb.13.8.5043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Van Houten J. V., Newlon C. S. Mutational analysis of the consensus sequence of a replication origin from yeast chromosome III. Mol Cell Biol. 1990 Aug;10(8):3917–3925. doi: 10.1128/mcb.10.8.3917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Wang T. S. Eukaryotic DNA polymerases. Annu Rev Biochem. 1991;60:513–552. doi: 10.1146/annurev.bi.60.070191.002501. [DOI] [PubMed] [Google Scholar]
  64. Waseem N. H., Labib K., Nurse P., Lane D. P. Isolation and analysis of the fission yeast gene encoding polymerase delta accessory protein PCNA. EMBO J. 1992 Dec;11(13):5111–5120. doi: 10.1002/j.1460-2075.1992.tb05618.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Wohlgemuth J. G., Bulboaca G. H., Moghadam M., Caddle M. S., Calos M. P. Physical mapping of origins of replication in the fission yeast Schizosaccharomyces pombe. Mol Biol Cell. 1994 Aug;5(8):839–849. doi: 10.1091/mbc.5.8.839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Yoon H. J., Campbell J. L. The CDC7 protein of Saccharomyces cerevisiae is a phosphoprotein that contains protein kinase activity. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3574–3578. doi: 10.1073/pnas.88.9.3574. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES