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. 1997 Aug;17(8):4654–4666. doi: 10.1128/mcb.17.8.4654

Structure-function analysis of the Saccharomyces cerevisiae G1 cyclin Cln2.

K N Huang 1, S A Odinsky 1, F R Cross 1
PMCID: PMC232318  PMID: 9234722

Abstract

We have generated 50 new alleles of the yeast CLN2 gene by using site-directed mutagenesis. With the recently obtained crystal structure of cyclin A as a guide, a peptide linker sequence was inserted at 13 sites within the cyclin box of Cln2 to determine if the architecture of Cln2 is similar to that of cyclin A. Linkers inserted in what are predicted to be helices 1, 2, 3, and 5 of the cyclin box resulted in nonfunctional Cln2 molecules. Linkers inserted between these putative helix sites and in the region believed to contain a fourth helix did not have significant effects upon Cln2 function. A series of deletions in the region between the third and fifth helices indicate that the putative fourth helix may lie at the C-terminal end of this region yet is not essential for function. Two residues that are predicted to form a buried salt bridge important for interaction of two helices of the cyclin box were also mutated, and an additional set of 31 mutant alleles was generated by clustered-charge-to-alanine scanning mutagenesis. All of the mutant CLN2 alleles made in this study were tested in a variety of genetic and functional assays previously demonstrated to differentiate specific cyclin functions. Some alleles demonstrated restricted patterns of defects, suggesting that these mutations may interfere with specific aspects of Cln2 function.

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Selected References

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

  1. Bazan J. F. Helical fold prediction for the cyclin box. Proteins. 1996 Jan;24(1):1–17. doi: 10.1002/(SICI)1097-0134(199601)24:1<1::AID-PROT1>3.0.CO;2-O. [DOI] [PubMed] [Google Scholar]
  2. Bennett W. F., Paoni N. F., Keyt B. A., Botstein D., Jones A. J., Presta L., Wurm F. M., Zoller M. J. High resolution analysis of functional determinants on human tissue-type plasminogen activator. J Biol Chem. 1991 Mar 15;266(8):5191–5201. [PubMed] [Google Scholar]
  3. Brown N. R., Noble M. E., Endicott J. A., Garman E. F., Wakatsuki S., Mitchell E., Rasmussen B., Hunt T., Johnson L. N. The crystal structure of cyclin A. Structure. 1995 Nov 15;3(11):1235–1247. doi: 10.1016/s0969-2126(01)00259-3. [DOI] [PubMed] [Google Scholar]
  4. Chang F., Herskowitz I. Identification of a gene necessary for cell cycle arrest by a negative growth factor of yeast: FAR1 is an inhibitor of a G1 cyclin, CLN2. Cell. 1990 Nov 30;63(5):999–1011. doi: 10.1016/0092-8674(90)90503-7. [DOI] [PubMed] [Google Scholar]
  5. Cross F. R. DAF1, a mutant gene affecting size control, pheromone arrest, and cell cycle kinetics of Saccharomyces cerevisiae. Mol Cell Biol. 1988 Nov;8(11):4675–4684. doi: 10.1128/mcb.8.11.4675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cross F. R. Starting the cell cycle: what's the point? Curr Opin Cell Biol. 1995 Dec;7(6):790–797. doi: 10.1016/0955-0674(95)80062-x. [DOI] [PubMed] [Google Scholar]
  7. Cunningham B. C., Wells J. A. High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science. 1989 Jun 2;244(4908):1081–1085. doi: 10.1126/science.2471267. [DOI] [PubMed] [Google Scholar]
  8. Di Como C. J., Chang H., Arndt K. T. Activation of CLN1 and CLN2 G1 cyclin gene expression by BCK2. Mol Cell Biol. 1995 Apr;15(4):1835–1846. doi: 10.1128/mcb.15.4.1835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dirick L., Böhm T., Nasmyth K. Roles and regulation of Cln-Cdc28 kinases at the start of the cell cycle of Saccharomyces cerevisiae. EMBO J. 1995 Oct 2;14(19):4803–4813. doi: 10.1002/j.1460-2075.1995.tb00162.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Epstein C. B., Cross F. R. Genes that can bypass the CLN requirement for Saccharomyces cerevisiae cell cycle START. Mol Cell Biol. 1994 Mar;14(3):2041–2047. doi: 10.1128/mcb.14.3.2041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hassett D. E., Condit R. C. Targeted construction of temperature-sensitive mutations in vaccinia virus by replacing clustered charged residues with alanine. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4554–4558. doi: 10.1073/pnas.91.10.4554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Horton R. M., Hunt H. D., Ho S. N., Pullen J. K., Pease L. R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene. 1989 Apr 15;77(1):61–68. doi: 10.1016/0378-1119(89)90359-4. [DOI] [PubMed] [Google Scholar]
  13. Jeffrey P. D., Russo A. A., Polyak K., Gibbs E., Hurwitz J., Massagué J., Pavletich N. P. Mechanism of CDK activation revealed by the structure of a cyclinA-CDK2 complex. Nature. 1995 Jul 27;376(6538):313–320. doi: 10.1038/376313a0. [DOI] [PubMed] [Google Scholar]
  14. Kim K. K., Chamberlin H. M., Morgan D. O., Kim S. H. Three-dimensional structure of human cyclin H, a positive regulator of the CDK-activating kinase. Nat Struct Biol. 1996 Oct;3(10):849–855. doi: 10.1038/nsb1096-849. [DOI] [PubMed] [Google Scholar]
  15. Kobayashi H., Stewart E., Poon R., Adamczewski J. P., Gannon J., Hunt T. Identification of the domains in cyclin A required for binding to, and activation of, p34cdc2 and p32cdk2 protein kinase subunits. Mol Biol Cell. 1992 Nov;3(11):1279–1294. doi: 10.1091/mbc.3.11.1279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Koch C., Nasmyth K. Cell cycle regulated transcription in yeast. Curr Opin Cell Biol. 1994 Jun;6(3):451–459. doi: 10.1016/0955-0674(94)90039-6. [DOI] [PubMed] [Google Scholar]
  17. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Levine K., Huang K., Cross F. R. Saccharomyces cerevisiae G1 cyclins differ in their intrinsic functional specificities. Mol Cell Biol. 1996 Dec;16(12):6794–6803. doi: 10.1128/mcb.16.12.6794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Measday V., Moore L., Ogas J., Tyers M., Andrews B. The PCL2 (ORFD)-PHO85 cyclin-dependent kinase complex: a cell cycle regulator in yeast. Science. 1994 Nov 25;266(5189):1391–1395. doi: 10.1126/science.7973731. [DOI] [PubMed] [Google Scholar]
  20. Mendenhall M. D. An inhibitor of p34CDC28 protein kinase activity from Saccharomyces cerevisiae. Science. 1993 Jan 8;259(5092):216–219. doi: 10.1126/science.8421781. [DOI] [PubMed] [Google Scholar]
  21. Nash R., Tokiwa G., Anand S., Erickson K., Futcher A. B. The WHI1+ gene of Saccharomyces cerevisiae tethers cell division to cell size and is a cyclin homolog. EMBO J. 1988 Dec 20;7(13):4335–4346. doi: 10.1002/j.1460-2075.1988.tb03332.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ogas J., Andrews B. J., Herskowitz I. Transcriptional activation of CLN1, CLN2, and a putative new G1 cyclin (HCS26) by SWI4, a positive regulator of G1-specific transcription. Cell. 1991 Sep 6;66(5):1015–1026. doi: 10.1016/0092-8674(91)90445-5. [DOI] [PubMed] [Google Scholar]
  23. Richardson H. E., Wittenberg C., Cross F., Reed S. I. An essential G1 function for cyclin-like proteins in yeast. Cell. 1989 Dec 22;59(6):1127–1133. doi: 10.1016/0092-8674(89)90768-x. [DOI] [PubMed] [Google Scholar]
  24. Schwob E., Böhm T., Mendenhall M. D., Nasmyth K. The B-type cyclin kinase inhibitor p40SIC1 controls the G1 to S transition in S. cerevisiae. Cell. 1994 Oct 21;79(2):233–244. doi: 10.1016/0092-8674(94)90193-7. [DOI] [PubMed] [Google Scholar]
  25. Schwob E., Nasmyth K. CLB5 and CLB6, a new pair of B cyclins involved in DNA replication in Saccharomyces cerevisiae. Genes Dev. 1993 Jul;7(7A):1160–1175. doi: 10.1101/gad.7.7a.1160. [DOI] [PubMed] [Google Scholar]
  26. Silence K., Hartmann M., Gührs K. H., Gase A., Schlott B., Collen D., Lijnen H. R. Structure-function relationships in staphylokinase as revealed by "clustered charge to alanine" mutagenesis. J Biol Chem. 1995 Nov 10;270(45):27192–27198. doi: 10.1074/jbc.270.45.27192. [DOI] [PubMed] [Google Scholar]
  27. Stewart E., Kobayashi H., Harrison D., Hunt T. Destruction of Xenopus cyclins A and B2, but not B1, requires binding to p34cdc2. EMBO J. 1994 Feb 1;13(3):584–594. doi: 10.1002/j.1460-2075.1994.tb06296.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Stuart D., Wittenberg C. CLN3, not positive feedback, determines the timing of CLN2 transcription in cycling cells. Genes Dev. 1995 Nov 15;9(22):2780–2794. doi: 10.1101/gad.9.22.2780. [DOI] [PubMed] [Google Scholar]
  29. Tyers M. The cyclin-dependent kinase inhibitor p40SIC1 imposes the requirement for Cln G1 cyclin function at Start. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7772–7776. doi: 10.1073/pnas.93.15.7772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ueshima S., Holvoet P., Lijnen H. R., Nelles L., Seghers V., Collen D. Expression and characterization of clustered charge-to-alanine mutants of low M(r) single-chain urokinase-type plasminogen activator. Thromb Haemost. 1994 Jan;71(1):134–140. [PubMed] [Google Scholar]

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