Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Sep;15(9):5165–5172. doi: 10.1128/mcb.15.9.5165

Replication of centromere II of Schizosaccharomyces pombe.

J G Smith 1, M S Caddle 1, G H Bulboaca 1, J G Wohlgemuth 1, M Baum 1, L Clarke 1, M P Calos 1
PMCID: PMC230763  PMID: 7651433

Abstract

The centromeric DNAs of Schizosaccharomyces pombe chromosomes resemble those of higher eukaryotes in being large and composed predominantly of repeated sequences. To begin a detailed analysis of the mode of replication of a complex centromere, we examined whether any sequences within S. pombe centromere II (cen2) have the ability to mediate autonomous replication. We found a high density of segments with such activity, including at least eight different regions comprising most of the repeated and unique centromeric DNA elements. A physical mapping analysis using two-dimensional gels showed that autonomous replication initiated within the S. pombe sequences in each plasmid. A two-dimensional gel analysis of replication on the chromosomes revealed that the K and L repeat elements, which occur in multiple copies at all three centromeres and comprise approximately 70% of total centromeric DNA mass in S. pombe, are both sites of replication initiation. In contrast, the unique cen2 central core, which contains multiple segments that can support autonomous replication, appears to be repressed for initiation on the chromosome. We discuss the implications of these findings for our understanding of DNA replication and centromere function.

Full Text

The Full Text of this article is available as a PDF (782.1 KB).

Selected References

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

  1. Baum M., Ngan V. K., Clarke L. The centromeric K-type repeat and the central core are together sufficient to establish a functional Schizosaccharomyces pombe centromere. Mol Biol Cell. 1994 Jul;5(7):747–761. doi: 10.1091/mbc.5.7.747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brewer B. J., Fangman W. L. Initiation preference at a yeast origin of replication. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3418–3422. doi: 10.1073/pnas.91.8.3418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brewer B. J., Fangman W. L. The localization of replication origins on ARS plasmids in S. cerevisiae. Cell. 1987 Nov 6;51(3):463–471. doi: 10.1016/0092-8674(87)90642-8. [DOI] [PubMed] [Google Scholar]
  4. Brewer B. J., Lockshon D., Fangman W. L. The arrest of replication forks in the rDNA of yeast occurs independently of transcription. Cell. 1992 Oct 16;71(2):267–276. doi: 10.1016/0092-8674(92)90355-g. [DOI] [PubMed] [Google Scholar]
  5. Caddle M. S., Calos M. P. Analysis of the autonomous replication behavior in human cells of the dihydrofolate reductase putative chromosomal origin of replication. Nucleic Acids Res. 1992 Nov 25;20(22):5971–5978. doi: 10.1093/nar/20.22.5971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Chikashige Y., Kinoshita N., Nakaseko Y., Matsumoto T., Murakami S., Niwa O., Yanagida M. Composite motifs and repeat symmetry in S. pombe centromeres: direct analysis by integration of NotI restriction sites. Cell. 1989 Jun 2;57(5):739–751. doi: 10.1016/0092-8674(89)90789-7. [DOI] [PubMed] [Google Scholar]
  9. Clarke L., Baum M. P. Functional analysis of a centromere from fission yeast: a role for centromere-specific repeated DNA sequences. Mol Cell Biol. 1990 May;10(5):1863–1872. doi: 10.1128/mcb.10.5.1863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Clarke L., Baum M., Marschall L. G., Ngan V. K., Steiner N. C. Structure and function of Schizosaccharomyces pombe centromeres. Cold Spring Harb Symp Quant Biol. 1993;58:687–695. doi: 10.1101/sqb.1993.058.01.076. [DOI] [PubMed] [Google Scholar]
  11. Clarke L., Carbon J. The structure and function of yeast centromeres. Annu Rev Genet. 1985;19:29–55. doi: 10.1146/annurev.ge.19.120185.000333. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Dershowitz A., Newlon C. S. The effect on chromosome stability of deleting replication origins. Mol Cell Biol. 1993 Jan;13(1):391–398. doi: 10.1128/mcb.13.1.391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Deshpande A. M., Newlon C. S. The ARS consensus sequence is required for chromosomal origin function in Saccharomyces cerevisiae. Mol Cell Biol. 1992 Oct;12(10):4305–4313. doi: 10.1128/mcb.12.10.4305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dubey D. D., Davis L. R., Greenfeder S. A., Ong L. Y., Zhu J. G., Broach J. R., Newlon C. S., Huberman J. A. Evidence suggesting that the ARS elements associated with silencers of the yeast mating-type locus HML do not function as chromosomal DNA replication origins. Mol Cell Biol. 1991 Oct;11(10):5346–5355. doi: 10.1128/mcb.11.10.5346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Fishel B., Amstutz H., Baum M., Carbon J., Clarke L. Structural organization and functional analysis of centromeric DNA in the fission yeast Schizosaccharomyces pombe. Mol Cell Biol. 1988 Feb;8(2):754–763. doi: 10.1128/mcb.8.2.754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Greenfeder S. A., Newlon C. S. A replication map of a 61-kb circular derivative of Saccharomyces cerevisiae chromosome III. Mol Biol Cell. 1992 Sep;3(9):999–1013. doi: 10.1091/mbc.3.9.999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hahnenberger K. M., Carbon J., Clarke L. Identification of DNA regions required for mitotic and meiotic functions within the centromere of Schizosaccharomyces pombe chromosome I. Mol Cell Biol. 1991 Apr;11(4):2206–2215. doi: 10.1128/mcb.11.4.2206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Heinzel S. S., Krysan P. J., Tran C. T., Calos M. P. Autonomous DNA replication in human cells is affected by the size and the source of the DNA. Mol Cell Biol. 1991 Apr;11(4):2263–2272. doi: 10.1128/mcb.11.4.2263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hsiao C. L., Carbon J. High-frequency transformation of yeast by plasmids containing the cloned yeast ARG4 gene. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3829–3833. doi: 10.1073/pnas.76.8.3829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Huberman J. A., Spotila L. D., Nawotka K. A., el-Assouli S. M., Davis L. R. The in vivo replication origin of the yeast 2 microns plasmid. Cell. 1987 Nov 6;51(3):473–481. doi: 10.1016/0092-8674(87)90643-x. [DOI] [PubMed] [Google Scholar]
  23. Krysan P. J., Calos M. P. Replication initiates at multiple locations on an autonomously replicating plasmid in human cells. Mol Cell Biol. 1991 Mar;11(3):1464–1472. doi: 10.1128/mcb.11.3.1464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Krysan P. J., Haase S. B., Calos M. P. Isolation of human sequences that replicate autonomously in human cells. Mol Cell Biol. 1989 Mar;9(3):1026–1033. doi: 10.1128/mcb.9.3.1026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Krysan P. J., Smith J. G., Calos M. P. Autonomous replication in human cells of multimers of specific human and bacterial DNA sequences. Mol Cell Biol. 1993 May;13(5):2688–2696. doi: 10.1128/mcb.13.5.2688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Linskens M. H., Huberman J. A. Organization of replication of ribosomal DNA in Saccharomyces cerevisiae. Mol Cell Biol. 1988 Nov;8(11):4927–4935. doi: 10.1128/mcb.8.11.4927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Marschall L. G., Clarke L. A novel cis-acting centromeric DNA element affects S. pombe centromeric chromatin structure at a distance. J Cell Biol. 1995 Feb;128(4):445–454. doi: 10.1083/jcb.128.4.445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Maundrell K., Wright A. P., Piper M., Shall S. Evaluation of heterologous ARS activity in S. cerevisiae using cloned DNA from S. pombe. Nucleic Acids Res. 1985 May 24;13(10):3711–3722. doi: 10.1093/nar/13.10.3711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Murakami S., Matsumoto T., Niwa O., Yanagida M. Structure of the fission yeast centromere cen3: direct analysis of the reiterated inverted region. Chromosoma. 1991 Dec;101(4):214–221. doi: 10.1007/BF00365153. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Newlon C. S., Lipchitz L. R., Collins I., Deshpande A., Devenish R. J., Green R. P., Klein H. L., Palzkill T. G., Ren R. B., Synn S. Analysis of a circular derivative of Saccharomyces cerevisiae chromosome III: a physical map and identification and location of ARS elements. Genetics. 1991 Oct;129(2):343–357. doi: 10.1093/genetics/129.2.343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. Polizzi C., Clarke L. The chromatin structure of centromeres from fission yeast: differentiation of the central core that correlates with function. J Cell Biol. 1991 Jan;112(2):191–201. doi: 10.1083/jcb.112.2.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Prentice H. L. High efficiency transformation of Schizosaccharomyces pombe by electroporation. Nucleic Acids Res. 1992 Feb 11;20(3):621–621. doi: 10.1093/nar/20.3.621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Steiner N. C., Clarke L. A novel epigenetic effect can alter centromere function in fission yeast. Cell. 1994 Dec 2;79(5):865–874. doi: 10.1016/0092-8674(94)90075-2. [DOI] [PubMed] [Google Scholar]
  39. Steiner N. C., Hahnenberger K. M., Clarke L. Centromeres of the fission yeast Schizosaccharomyces pombe are highly variable genetic loci. Mol Cell Biol. 1993 Aug;13(8):4578–4587. doi: 10.1128/mcb.13.8.4578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Stinchcomb D. T., Struhl K., Davis R. W. Isolation and characterisation of a yeast chromosomal replicator. Nature. 1979 Nov 1;282(5734):39–43. doi: 10.1038/282039a0. [DOI] [PubMed] [Google Scholar]
  41. Struhl K., Stinchcomb D. T., Scherer S., Davis R. W. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1035–1039. doi: 10.1073/pnas.76.3.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Takahashi K., Murakami S., Chikashige Y., Funabiki H., Niwa O., Yanagida M. A low copy number central sequence with strict symmetry and unusual chromatin structure in fission yeast centromere. Mol Biol Cell. 1992 Jul;3(7):819–835. doi: 10.1091/mbc.3.7.819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Willard H. F. Centromeres of mammalian chromosomes. Trends Genet. 1990 Dec;6(12):410–416. doi: 10.1016/0168-9525(90)90302-m. [DOI] [PubMed] [Google Scholar]
  45. Williamson D. H. The yeast ARS element, six years on: a progress report. Yeast. 1985 Sep;1(1):1–14. doi: 10.1002/yea.320010102. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Wright A. P., Maundrell K., Shall S. Transformation of Schizosaccharomyces pombe by non-homologous, unstable integration of plasmids in the genome. Curr Genet. 1986;10(7):503–508. doi: 10.1007/BF00447383. [DOI] [PubMed] [Google Scholar]
  48. Zhu J., Brun C., Kurooka H., Yanagida M., Huberman J. A. Identification and characterization of a complex chromosomal replication origin in Schizosaccharomyces pombe. Chromosoma. 1992;102(1 Suppl):S7–16. doi: 10.1007/BF02451780. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES