Skip to main content
NCBI home page
Genetics logoLink to Genetics
. 1999 Apr;151(4):1445–1457. doi: 10.1093/genetics/151.4.1445

The role of nucleotide binding and hydrolysis in the function of the fission yeast cdc18(+) gene product.

D DeRyckere 1, C L Smith 1, G S Martin 1
PMCID: PMC1460557  PMID: 10101168

Abstract

The fission yeast cdc18(+) gene is required for both initiation of DNA replication and the mitotic checkpoint that normally inhibits mitosis in the absence of DNA replication. The cdc18(+) gene product contains conserved Walker A and B box motifs. Studies of other ATPases have shown that these motifs are required for nucleotide binding and hydrolysis, respectively. We have observed that mutant strains in which either of these motifs is disrupted are inviable. The effects of these mutations were examined by determining the phenotypes of mutant strains following depletion of complementing wild-type Cdc18. In both synchronous and asynchronous cultures, the nucleotide-hydrolysis motif mutant (DE286AA) arrests with a 1C-2C DNA content, and thus exhibits no obvious defects in entry into S phase or in the mitotic checkpoint. In contrast, in cultures synchronized by hydroxyurea arrest and release, the nucleotide-binding motif mutant (K205A) exhibits the null phenotype, with 1C and <1C DNA content, indicating a block in entry into S phase and loss of checkpoint control. In asynchronous cultures this mutant exhibits a mixed phenotype: a percentage of the population displays the null phenotype, while the remaining fraction arrests with a 2C DNA content. Thus, the phenotype exhibited by the K205A mutant is dependent on the cell-cycle position at which wild-type Cdc18 is depleted. These data indicate that both nucleotide binding and hydrolysis are required for Cdc18 function. In addition, the difference in the phenotypes exhibited by the nucleotide-binding and hydrolysis motif mutants is consistent with a two-step model for Cdc18 function in which nucleotide binding and hydrolysis are required for distinct aspects of Cdc18 function that may be executed at different points in the cell cycle.

Full Text

The Full Text of this article is available as a PDF (279.3 KB).

Selected References

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

  1. Aparicio O. M., Weinstein D. M., Bell S. P. Components and dynamics of DNA replication complexes in S. cerevisiae: redistribution of MCM proteins and Cdc45p during S phase. Cell. 1997 Oct 3;91(1):59–69. doi: 10.1016/s0092-8674(01)80009-x. [DOI] [PubMed] [Google Scholar]
  2. Bell S. P., Mitchell J., Leber J., Kobayashi R., Stillman B. The multidomain structure of Orc1p reveals similarity to regulators of DNA replication and transcriptional silencing. Cell. 1995 Nov 17;83(4):563–568. doi: 10.1016/0092-8674(95)90096-9. [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. Carpenter P. B., Mueller P. R., Dunphy W. G. Role for a Xenopus Orc2-related protein in controlling DNA replication. Nature. 1996 Jan 25;379(6563):357–360. doi: 10.1038/379357a0. [DOI] [PubMed] [Google Scholar]
  5. Chong J. P., Mahbubani H. M., Khoo C. Y., Blow J. J. Purification of an MCM-containing complex as a component of the DNA replication licensing system. Nature. 1995 Jun 1;375(6530):418–421. doi: 10.1038/375418a0. [DOI] [PubMed] [Google Scholar]
  6. Cocker J. H., Piatti S., Santocanale C., Nasmyth K., Diffley J. F. An essential role for the Cdc6 protein in forming the pre-replicative complexes of budding yeast. Nature. 1996 Jan 11;379(6561):180–182. doi: 10.1038/379180a0. [DOI] [PubMed] [Google Scholar]
  7. Coleman T. R., Carpenter P. B., Dunphy W. G. The Xenopus Cdc6 protein is essential for the initiation of a single round of DNA replication in cell-free extracts. Cell. 1996 Oct 4;87(1):53–63. doi: 10.1016/s0092-8674(00)81322-7. [DOI] [PubMed] [Google Scholar]
  8. Diffley J. F., Cocker J. H., Dowell S. J., Rowley A. Two steps in the assembly of complexes at yeast replication origins in vivo. Cell. 1994 Jul 29;78(2):303–316. doi: 10.1016/0092-8674(94)90299-2. [DOI] [PubMed] [Google Scholar]
  9. Diffley J. F., Cocker J. H. Protein-DNA interactions at a yeast replication origin. Nature. 1992 May 14;357(6374):169–172. doi: 10.1038/357169a0. [DOI] [PubMed] [Google Scholar]
  10. Donovan S., Harwood J., Drury L. S., Diffley J. F. Cdc6p-dependent loading of Mcm proteins onto pre-replicative chromatin in budding yeast. Proc Natl Acad Sci U S A. 1997 May 27;94(11):5611–5616. doi: 10.1073/pnas.94.11.5611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ehrenhofer-Murray A. E., Gossen M., Pak D. T., Botchan M. R., Rine J. Separation of origin recognition complex functions by cross-species complementation. Science. 1995 Dec 8;270(5242):1671–1674. doi: 10.1126/science.270.5242.1671. [DOI] [PubMed] [Google Scholar]
  12. Fox C. A., Loo S., Dillin A., Rine J. The origin recognition complex has essential functions in transcriptional silencing and chromosomal replication. Genes Dev. 1995 Apr 15;9(8):911–924. doi: 10.1101/gad.9.8.911. [DOI] [PubMed] [Google Scholar]
  13. Fry D. C., Kuby S. A., Mildvan A. S. ATP-binding site of adenylate kinase: mechanistic implications of its homology with ras-encoded p21, F1-ATPase, and other nucleotide-binding proteins. Proc Natl Acad Sci U S A. 1986 Feb;83(4):907–911. doi: 10.1073/pnas.83.4.907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gavin K. A., Hidaka M., Stillman B. Conserved initiator proteins in eukaryotes. Science. 1995 Dec 8;270(5242):1667–1671. doi: 10.1126/science.270.5242.1667. [DOI] [PubMed] [Google Scholar]
  15. Gorbalenya A. E., Koonin E. V., Donchenko A. P., Blinov V. M. Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Nucleic Acids Res. 1989 Jun 26;17(12):4713–4730. doi: 10.1093/nar/17.12.4713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gossen M., Pak D. T., Hansen S. K., Acharya J. K., Botchan M. R. A Drosophila homolog of the yeast origin recognition complex. Science. 1995 Dec 8;270(5242):1674–1677. doi: 10.1126/science.270.5242.1674. [DOI] [PubMed] [Google Scholar]
  17. Grallert B., Nurse P. The ORC1 homolog orp1 in fission yeast plays a key role in regulating onset of S phase. Genes Dev. 1996 Oct 15;10(20):2644–2654. doi: 10.1101/gad.10.20.2644. [DOI] [PubMed] [Google Scholar]
  18. Haber L. T., Walker G. C. Altering the conserved nucleotide binding motif in the Salmonella typhimurium MutS mismatch repair protein affects both its ATPase and mismatch binding activities. EMBO J. 1991 Sep;10(9):2707–2715. doi: 10.1002/j.1460-2075.1991.tb07815.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hennessy K. M., Clark C. D., Botstein D. Subcellular localization of yeast CDC46 varies with the cell cycle. Genes Dev. 1990 Dec;4(12B):2252–2263. doi: 10.1101/gad.4.12b.2252. [DOI] [PubMed] [Google Scholar]
  20. Hennessy K. M., Lee A., Chen E., Botstein D. A group of interacting yeast DNA replication genes. Genes Dev. 1991 Jun;5(6):958–969. doi: 10.1101/gad.5.6.958. [DOI] [PubMed] [Google Scholar]
  21. Hodgman T. C. A new superfamily of replicative proteins. Nature. 1988 May 5;333(6168):22–23. doi: 10.1038/333022b0. [DOI] [PubMed] [Google Scholar]
  22. Jallepalli P. V., Brown G. W., Muzi-Falconi M., Tien D., Kelly T. J. Regulation of the replication initiator protein p65cdc18 by CDK phosphorylation. Genes Dev. 1997 Nov 1;11(21):2767–2779. doi: 10.1101/gad.11.21.2767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Keeney J. B., Boeke J. D. Efficient targeted integration at leu1-32 and ura4-294 in Schizosaccharomyces pombe. Genetics. 1994 Mar;136(3):849–856. doi: 10.1093/genetics/136.3.849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Klemm R. D., Austin R. J., Bell S. P. Coordinate binding of ATP and origin DNA regulates the ATPase activity of the origin recognition complex. Cell. 1997 Feb 21;88(4):493–502. doi: 10.1016/s0092-8674(00)81889-9. [DOI] [PubMed] [Google Scholar]
  26. Koonin E. V. A common set of conserved motifs in a vast variety of putative nucleic acid-dependent ATPases including MCM proteins involved in the initiation of eukaryotic DNA replication. Nucleic Acids Res. 1993 Jun 11;21(11):2541–2547. doi: 10.1093/nar/21.11.2541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kubota Y., Mimura S., Nishimoto S., Takisawa H., Nojima H. Identification of the yeast MCM3-related protein as a component of Xenopus DNA replication licensing factor. Cell. 1995 May 19;81(4):601–609. doi: 10.1016/0092-8674(95)90081-0. [DOI] [PubMed] [Google Scholar]
  28. Lopez-Girona A., Mondesert O., Leatherwood J., Russell P. Negative regulation of Cdc18 DNA replication protein by Cdc2. Mol Biol Cell. 1998 Jan;9(1):63–73. doi: 10.1091/mbc.9.1.63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Madine M. A., Khoo C. Y., Mills A. D., Laskey R. A. MCM3 complex required for cell cycle regulation of DNA replication in vertebrate cells. Nature. 1995 Jun 1;375(6530):421–424. doi: 10.1038/375421a0. [DOI] [PubMed] [Google Scholar]
  30. Maine G. T., Sinha P., Tye B. K. Mutants of S. cerevisiae defective in the maintenance of minichromosomes. Genetics. 1984 Mar;106(3):365–385. doi: 10.1093/genetics/106.3.365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Maiorano D., Van Assendelft G. B., Kearsey S. E. Fission yeast cdc21, a member of the MCM protein family, is required for onset of S phase and is located in the nucleus throughout the cell cycle. EMBO J. 1996 Feb 15;15(4):861–872. [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Muzi Falconi M., Brown G. W., Kelly T. J. cdc18+ regulates initiation of DNA replication in Schizosaccharomyces pombe. Proc Natl Acad Sci U S A. 1996 Feb 20;93(4):1566–1570. doi: 10.1073/pnas.93.4.1566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Muzi-Falconi M., Brown G. W., Kelly T. J. Controlling initiation during the cell cycle. DNA replication. Curr Biol. 1996 Mar 1;6(3):229–233. doi: 10.1016/s0960-9822(02)00464-5. [DOI] [PubMed] [Google Scholar]
  35. Muzi-Falconi M., Kelly T. J. Orp1, a member of the Cdc18/Cdc6 family of S-phase regulators, is homologous to a component of the origin recognition complex. Proc Natl Acad Sci U S A. 1995 Dec 19;92(26):12475–12479. doi: 10.1073/pnas.92.26.12475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nishitani H., Nurse P. p65cdc18 plays a major role controlling the initiation of DNA replication in fission yeast. Cell. 1995 Nov 3;83(3):397–405. doi: 10.1016/0092-8674(95)90117-5. [DOI] [PubMed] [Google Scholar]
  37. Pai E. F., Krengel U., Petsko G. A., Goody R. S., Kabsch W., Wittinghofer A. Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis. EMBO J. 1990 Aug;9(8):2351–2359. doi: 10.1002/j.1460-2075.1990.tb07409.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Pause A., Sonenberg N. Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A. EMBO J. 1992 Jul;11(7):2643–2654. doi: 10.1002/j.1460-2075.1992.tb05330.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Rowles A., Chong J. P., Brown L., Howell M., Evan G. I., Blow J. J. Interaction between the origin recognition complex and the replication licensing system in Xenopus. Cell. 1996 Oct 18;87(2):287–296. doi: 10.1016/s0092-8674(00)81346-x. [DOI] [PubMed] [Google Scholar]
  40. Rozen F., Pelletier J., Trachsel H., Sonenberg N. A lysine substitution in the ATP-binding site of eucaryotic initiation factor 4A abrogates nucleotide-binding activity. Mol Cell Biol. 1989 Sep;9(9):4061–4063. doi: 10.1128/mcb.9.9.4061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sinha P., Chang V., Tye B. K. A mutant that affects the function of autonomously replicating sequences in yeast. J Mol Biol. 1986 Dec 20;192(4):805–814. doi: 10.1016/0022-2836(86)90030-6. [DOI] [PubMed] [Google Scholar]
  42. Song J. M., Montelone B. A., Siede W., Friedberg E. C. Effects of multiple yeast rad3 mutant alleles on UV sensitivity, mutability, and mitotic recombination. J Bacteriol. 1990 Dec;172(12):6620–6630. doi: 10.1128/jb.172.12.6620-6630.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Story R. M., Steitz T. A. Structure of the recA protein-ADP complex. Nature. 1992 Jan 23;355(6358):374–376. doi: 10.1038/355374a0. [DOI] [PubMed] [Google Scholar]
  44. Takahara K., Bong M., Brevard R., Eddy R. L., Haley L. L., Sait S. J., Shows T. B., Hoffman G. G., Greenspan D. S. Mouse and human homologues of the yeast origin of replication recognition complex subunit ORC2 and chromosomal localization of the cognate human gene ORC2L. Genomics. 1996 Jan 1;31(1):119–122. doi: 10.1006/geno.1996.0018. [DOI] [PubMed] [Google Scholar]
  45. Tanaka T., Knapp D., Nasmyth K. Loading of an Mcm protein onto DNA replication origins is regulated by Cdc6p and CDKs. Cell. 1997 Aug 22;90(4):649–660. doi: 10.1016/s0092-8674(00)80526-7. [DOI] [PubMed] [Google Scholar]
  46. Walker J. E., Saraste M., Runswick M. J., Gay N. J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1(8):945–951. doi: 10.1002/j.1460-2075.1982.tb01276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. Weng Y., Czaplinski K., Peltz S. W. Genetic and biochemical characterization of mutations in the ATPase and helicase regions of the Upf1 protein. Mol Cell Biol. 1996 Oct;16(10):5477–5490. doi: 10.1128/mcb.16.10.5477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Yan H., Merchant A. M., Tye B. K. Cell cycle-regulated nuclear localization of MCM2 and MCM3, which are required for the initiation of DNA synthesis at chromosomal replication origins in yeast. Genes Dev. 1993 Nov;7(11):2149–2160. doi: 10.1101/gad.7.11.2149. [DOI] [PubMed] [Google Scholar]
  50. Zhu L. A., Weller S. K. The six conserved helicase motifs of the UL5 gene product, a component of the herpes simplex virus type 1 helicase-primase, are essential for its function. J Virol. 1992 Jan;66(1):469–479. doi: 10.1128/jvi.66.1.469-479.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES