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. 1991 Jul;11(7):3545–3553. doi: 10.1128/mcb.11.7.3545

In vivo characterization of the Saccharomyces cerevisiae centromere DNA element I, a binding site for the helix-loop-helix protein CPF1.

R Niedenthal 1, R Stoll 1, J H Hegemann 1
PMCID: PMC361097  PMID: 2046668

Abstract

The centromere DNA element I (CDEI) is an important component of Saccharomyces cerevisiae centromere DNA and carries the palindromic sequence CACRTG (R = purine) as a characteristic feature. In vivo, CDEI is bound by the helix-loop-helix protein CPF1. This article describes the in vivo analysis of all single-base-pair substitutions in CDEI in the centromere of an artificial chromosome and demonstrates the importance of the palindromic sequence for faithful chromosome segregation, supporting the notion that CPF1 binds as a dimer to this binding site. Mutational analysis of two conserved base pairs on the left and two nonconserved base pairs on the right of the CDEI palindrome revealed that these are also relevant for mitotic CEN function. Symmetrical mutations in either half-site of the palindrome affect centromere activity to a different extent, indicating nonidentical sequence requirements for binding by the CPF1 homodimer. Analysis of double point mutations in CDEI and in CDEIII, an additional centromere element, indicate synergistic effects between the DNA-protein complexes at these sites.

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Selected References

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

  1. Aggarwal A. K., Rodgers D. W., Drottar M., Ptashne M., Harrison S. C. Recognition of a DNA operator by the repressor of phage 434: a view at high resolution. Science. 1988 Nov 11;242(4880):899–907. doi: 10.1126/science.3187531. [DOI] [PubMed] [Google Scholar]
  2. Baker R. E., Fitzgerald-Hayes M., O'Brien T. C. Purification of the yeast centromere binding protein CP1 and a mutational analysis of its binding site. J Biol Chem. 1989 Jun 25;264(18):10843–10850. [PubMed] [Google Scholar]
  3. Baker R. E., Masison D. C. Isolation of the gene encoding the Saccharomyces cerevisiae centromere-binding protein CP1. Mol Cell Biol. 1990 Jun;10(6):2458–2467. doi: 10.1128/mcb.10.6.2458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beckmann H., Su L. K., Kadesch T. TFE3: a helix-loop-helix protein that activates transcription through the immunoglobulin enhancer muE3 motif. Genes Dev. 1990 Feb;4(2):167–179. doi: 10.1101/gad.4.2.167. [DOI] [PubMed] [Google Scholar]
  5. Bloom K. S., Carbon J. Yeast centromere DNA is in a unique and highly ordered structure in chromosomes and small circular minichromosomes. Cell. 1982 Jun;29(2):305–317. doi: 10.1016/0092-8674(82)90147-7. [DOI] [PubMed] [Google Scholar]
  6. Bram R. J., Kornberg R. D. Isolation of a Saccharomyces cerevisiae centromere DNA-binding protein, its human homolog, and its possible role as a transcription factor. Mol Cell Biol. 1987 Jan;7(1):403–409. doi: 10.1128/mcb.7.1.403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Braun T., Winter B., Bober E., Arnold H. H. Transcriptional activation domain of the muscle-specific gene-regulatory protein myf5. Nature. 1990 Aug 16;346(6285):663–665. doi: 10.1038/346663a0. [DOI] [PubMed] [Google Scholar]
  8. Cai M. J., Davis R. W. Purification of a yeast centromere-binding protein that is able to distinguish single base-pair mutations in its recognition site. Mol Cell Biol. 1989 Jun;9(6):2544–2550. doi: 10.1128/mcb.9.6.2544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cai M., Davis R. W. Yeast centromere binding protein CBF1, of the helix-loop-helix protein family, is required for chromosome stability and methionine prototrophy. Cell. 1990 May 4;61(3):437–446. doi: 10.1016/0092-8674(90)90525-j. [DOI] [PubMed] [Google Scholar]
  10. Carr C. S., Sharp P. A. A helix-loop-helix protein related to the immunoglobulin E box-binding proteins. Mol Cell Biol. 1990 Aug;10(8):4384–4388. doi: 10.1128/mcb.10.8.4384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chodosh L. A., Buratowski S., Sharp P. A. A yeast protein possesses the DNA-binding properties of the adenovirus major late transcription factor. Mol Cell Biol. 1989 Feb;9(2):820–822. doi: 10.1128/mcb.9.2.820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chodosh L. A., Carthew R. W., Morgan J. G., Crabtree G. R., Sharp P. A. The adenovirus major late transcription factor activates the rat gamma-fibrinogen promoter. Science. 1987 Oct 30;238(4827):684–688. doi: 10.1126/science.3672119. [DOI] [PubMed] [Google Scholar]
  13. Clarke L., Amstutz H., Fishel B., Carbon J. Analysis of centromeric DNA in the fission yeast Schizosaccharomyces pombe. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8253–8257. doi: 10.1073/pnas.83.21.8253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Clarke L., Carbon J. Isolation of a yeast centromere and construction of functional small circular chromosomes. Nature. 1980 Oct 9;287(5782):504–509. doi: 10.1038/287504a0. [DOI] [PubMed] [Google Scholar]
  15. Clarke L. Centromeres of budding and fission yeasts. Trends Genet. 1990 May;6(5):150–154. doi: 10.1016/0168-9525(90)90149-z. [DOI] [PubMed] [Google Scholar]
  16. Cottarel G., Shero J. H., Hieter P., Hegemann J. H. A 125-base-pair CEN6 DNA fragment is sufficient for complete meiotic and mitotic centromere functions in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Aug;9(8):3342–3349. doi: 10.1128/mcb.9.8.3342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Cumberledge S., Carbon J. Mutational analysis of meiotic and mitotic centromere function in Saccharomyces cerevisiae. Genetics. 1987 Oct;117(2):203–212. doi: 10.1093/genetics/117.2.203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Dorsman J. C., van Heeswijk W. C., Grivell L. A. Identification of two factors which bind to the upstream sequences of a number of nuclear genes coding for mitochondrial proteins and to genetic elements important for cell division in yeast. Nucleic Acids Res. 1988 Aug 11;16(15):7287–7301. doi: 10.1093/nar/16.15.7287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fitzgerald-Hayes M., Clarke L., Carbon J. Nucleotide sequence comparisons and functional analysis of yeast centromere DNAs. Cell. 1982 May;29(1):235–244. doi: 10.1016/0092-8674(82)90108-8. [DOI] [PubMed] [Google Scholar]
  20. Funk M., Hegemann J. H., Philippsen P. Chromatin digestion with restriction endonucleases reveals 150-160 bp of protected DNA in the centromere of chromosome XIV in Saccharomyces cerevisiae. Mol Gen Genet. 1989 Oct;219(1-2):153–160. doi: 10.1007/BF00261171. [DOI] [PubMed] [Google Scholar]
  21. Gaudet A., Fitzgerald-Hayes M. Alterations in the adenine-plus-thymine-rich region of CEN3 affect centromere function in Saccharomyces cerevisiae. Mol Cell Biol. 1987 Jan;7(1):68–75. doi: 10.1128/mcb.7.1.68. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gaudet A., Fitzgerald-Hayes M. Mutations in CEN3 cause aberrant chromosome segregation during meiosis in Saccharomyces cerevisiae. Genetics. 1989 Mar;121(3):477–489. doi: 10.1093/genetics/121.3.477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hall R. K., Taylor W. L. Transcription factor IIIA gene expression in Xenopus oocytes utilizes a transcription factor similar to the major late transcription factor. Mol Cell Biol. 1989 Nov;9(11):5003–5011. doi: 10.1128/mcb.9.11.5003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hegemann J. H., Pridmore R. D., Schneider R., Philippsen P. Mutations in the right boundary of Saccharomyces cerevisiae centromere 6 lead to nonfunctional or partially functional centromeres. Mol Gen Genet. 1986 Nov;205(2):305–311. doi: 10.1007/BF00430443. [DOI] [PubMed] [Google Scholar]
  25. Hegemann J. H., Shero J. H., Cottarel G., Philippsen P., Hieter P. Mutational analysis of centromere DNA from chromosome VI of Saccharomyces cerevisiae. Mol Cell Biol. 1988 Jun;8(6):2523–2535. doi: 10.1128/mcb.8.6.2523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hieter P., Mann C., Snyder M., Davis R. W. Mitotic stability of yeast chromosomes: a colony color assay that measures nondisjunction and chromosome loss. Cell. 1985 Feb;40(2):381–392. doi: 10.1016/0092-8674(85)90152-7. [DOI] [PubMed] [Google Scholar]
  27. Hieter P., Pridmore D., Hegemann J. H., Thomas M., Davis R. W., Philippsen P. Functional selection and analysis of yeast centromeric DNA. Cell. 1985 Oct;42(3):913–921. doi: 10.1016/0092-8674(85)90287-9. [DOI] [PubMed] [Google Scholar]
  28. Hope I. A., Struhl K. GCN4, a eukaryotic transcriptional activator protein, binds as a dimer to target DNA. EMBO J. 1987 Sep;6(9):2781–2784. doi: 10.1002/j.1460-2075.1987.tb02573.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Jiang W. D., Philippsen P. Purification of a protein binding to the CDEI subregion of Saccharomyces cerevisiae centromere DNA. Mol Cell Biol. 1989 Dec;9(12):5585–5593. doi: 10.1128/mcb.9.12.5585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Koudelka G. B., Harrison S. C., Ptashne M. Effect of non-contacted bases on the affinity of 434 operator for 434 repressor and Cro. 1987 Apr 30-May 6Nature. 326(6116):886–888. doi: 10.1038/326886a0. [DOI] [PubMed] [Google Scholar]
  32. Kramer W., Drutsa V., Jansen H. W., Kramer B., Pflugfelder M., Fritz H. J. The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 1984 Dec 21;12(24):9441–9456. doi: 10.1093/nar/12.24.9441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lassar A. B., Buskin J. N., Lockshon D., Davis R. L., Apone S., Hauschka S. D., Weintraub H. MyoD is a sequence-specific DNA binding protein requiring a region of myc homology to bind to the muscle creatine kinase enhancer. Cell. 1989 Sep 8;58(5):823–831. doi: 10.1016/0092-8674(89)90935-5. [DOI] [PubMed] [Google Scholar]
  34. Mellor J., Jiang W., Funk M., Rathjen J., Barnes C. A., Hinz T., Hegemann J. H., Philippsen P. CPF1, a yeast protein which functions in centromeres and promoters. EMBO J. 1990 Dec;9(12):4017–4026. doi: 10.1002/j.1460-2075.1990.tb07623.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Moncollin V., Stalder R., Verdier J. M., Sentenac A., Egly J. M. A yeast homolog of the human UEF stimulates transcription from the adenovirus 2 major late promoter in yeast and in mammalian cell-free systems. Nucleic Acids Res. 1990 Aug 25;18(16):4817–4823. doi: 10.1093/nar/18.16.4817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Murre C., McCaw P. S., Vaessin H., Caudy M., Jan L. Y., Jan Y. N., Cabrera C. V., Buskin J. N., Hauschka S. D., Lassar A. B. Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence. Cell. 1989 Aug 11;58(3):537–544. doi: 10.1016/0092-8674(89)90434-0. [DOI] [PubMed] [Google Scholar]
  37. Nakaseko Y., Adachi Y., Funahashi S., Niwa O., Yanagida M. Chromosome walking shows a highly homologous repetitive sequence present in all the centromere regions of fission yeast. EMBO J. 1986 May;5(5):1011–1021. doi: 10.1002/j.1460-2075.1986.tb04316.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Newlon C. S. Yeast chromosome replication and segregation. Microbiol Rev. 1988 Dec;52(4):568–601. doi: 10.1128/mr.52.4.568-601.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ng R., Carbon J. Mutational and in vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae. Mol Cell Biol. 1987 Dec;7(12):4522–4534. doi: 10.1128/mcb.7.12.4522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Ogawa N., Oshima Y. Functional domains of a positive regulatory protein, PHO4, for transcriptional control of the phosphatase regulon in Saccharomyces cerevisiae. Mol Cell Biol. 1990 May;10(5):2224–2236. doi: 10.1128/mcb.10.5.2224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Otwinowski Z., Schevitz R. W., Zhang R. G., Lawson C. L., Joachimiak A., Marmorstein R. Q., Luisi B. F., Sigler P. B. Crystal structure of trp repressor/operator complex at atomic resolution. Nature. 1988 Sep 22;335(6188):321–329. doi: 10.1038/335321a0. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Panzeri L., Landonio L., Stotz A., Philippsen P. Role of conserved sequence elements in yeast centromere DNA. EMBO J. 1985 Jul;4(7):1867–1874. doi: 10.1002/j.1460-2075.1985.tb03862.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sawadogo M., Roeder R. G. Interaction of a gene-specific transcription factor with the adenovirus major late promoter upstream of the TATA box region. Cell. 1985 Nov;43(1):165–175. doi: 10.1016/0092-8674(85)90021-2. [DOI] [PubMed] [Google Scholar]
  45. Scotto K. W., Kaulen H., Roeder R. G. Positive and negative regulation of the gene for transcription factor IIIA in Xenopus laevis oocytes. Genes Dev. 1989 May;3(5):651–662. doi: 10.1101/gad.3.5.651. [DOI] [PubMed] [Google Scholar]
  46. Shero J. H., Koval M., Spencer F., Palmer R. E., Hieter P., Koshland D. Analysis of chromosome segregation in Saccharomyces cerevisiae. Methods Enzymol. 1991;194:749–773. doi: 10.1016/0076-6879(91)94057-j. [DOI] [PubMed] [Google Scholar]
  47. Vogel K., Hörz W., Hinnen A. The two positively acting regulatory proteins PHO2 and PHO4 physically interact with PHO5 upstream activation regions. Mol Cell Biol. 1989 May;9(5):2050–2057. doi: 10.1128/mcb.9.5.2050. [DOI] [PMC free article] [PubMed] [Google Scholar]

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