Skip to main content
PMC home page
Genetics logoLink to Genetics
. 2001 Sep;159(1):35–45. doi: 10.1093/genetics/159.1.35

Roles for internal and flanking sequences in regulating the activity of mating-type-silencer-associated replication origins in Saccharomyces cerevisiae.

K Sharma 1, M Weinberger 1, J A Huberman 1
PMCID: PMC1461791  PMID: 11560885

Abstract

ARS301 and ARS302 are inactive replication origins located at the left end of budding yeast (Saccharomyces cerevisiae) chromosome III, where they are associated with the HML-E and -I silencers of the HML mating type cassette. Although they function as replication origins in plasmids, they do not serve as origins in their normal chromosomal locations, because they are programmed to fire so late in S phase that they are passively replicated by the replication fork from neighboring early-firing ARS305 before they have a chance to fire on their own. We asked whether the nucleotide sequences required for plasmid origin function of these silencer-associated chromosomally inactive origins differ from the sequences needed for plasmid origin function by nonsilencer-associated chromosomally active origins. We could not detect consistent differences in sequence requirements for the two types of origins. Next, we asked whether sequences within or flanking these origins are responsible for their chromosomal inactivity. Our results demonstrate that both flanking and internal sequences contribute to chromosomal inactivity, presumably by programming these origins to fire late in S phase. In ARS301, the function of the internal sequences determining chromosomal inactivity is dependent on the checkpoint proteins Mec1p and Rad53p.

Full Text

The Full Text of this article is available as a PDF (431.3 KB).

Selected References

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

  1. Allen J. B., Zhou Z., Siede W., Friedberg E. C., Elledge S. J. The SAD1/RAD53 protein kinase controls multiple checkpoints and DNA damage-induced transcription in yeast. Genes Dev. 1994 Oct 15;8(20):2401–2415. doi: 10.1101/gad.8.20.2401. [DOI] [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. Boeke J. D., Trueheart J., Natsoulis G., Fink G. R. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 1987;154:164–175. doi: 10.1016/0076-6879(87)54076-9. [DOI] [PubMed] [Google Scholar]
  4. Bousset K., Diffley J. F. The Cdc7 protein kinase is required for origin firing during S phase. Genes Dev. 1998 Feb 15;12(4):480–490. doi: 10.1101/gad.12.4.480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brand A. H., Micklem G., Nasmyth K. A yeast silencer contains sequences that can promote autonomous plasmid replication and transcriptional activation. Cell. 1987 Dec 4;51(5):709–719. doi: 10.1016/0092-8674(87)90094-8. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Broach J. R., Li Y. Y., Feldman J., Jayaram M., Abraham J., Nasmyth K. A., Hicks J. B. Localization and sequence analysis of yeast origins of DNA replication. Cold Spring Harb Symp Quant Biol. 1983;47(Pt 2):1165–1173. doi: 10.1101/sqb.1983.047.01.132. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Ferguson B. M., Fangman W. L. A position effect on the time of replication origin activation in yeast. Cell. 1992 Jan 24;68(2):333–339. doi: 10.1016/0092-8674(92)90474-q. [DOI] [PubMed] [Google Scholar]
  11. Friedman K. L., Brewer B. J., Fangman W. L. Replication profile of Saccharomyces cerevisiae chromosome VI. Genes Cells. 1997 Nov;2(11):667–678. doi: 10.1046/j.1365-2443.1997.1520350.x. [DOI] [PubMed] [Google Scholar]
  12. Huang R. Y., Kowalski D. A DNA unwinding element and an ARS consensus comprise a replication origin within a yeast chromosome. EMBO J. 1993 Dec;12(12):4521–4531. doi: 10.1002/j.1460-2075.1993.tb06141.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Huang R. Y., Kowalski D. Multiple DNA elements in ARS305 determine replication origin activity in a yeast chromosome. Nucleic Acids Res. 1996 Mar 1;24(5):816–823. doi: 10.1093/nar/24.5.816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Hurst S. T., Rivier D. H. Identification of a compound origin of replication at the HMR-E locus in Saccharomyces cerevisiae. J Biol Chem. 1999 Feb 12;274(7):4155–4159. doi: 10.1074/jbc.274.7.4155. [DOI] [PubMed] [Google Scholar]
  17. Laurenson P., Rine J. Silencers, silencing, and heritable transcriptional states. Microbiol Rev. 1992 Dec;56(4):543–560. doi: 10.1128/mr.56.4.543-560.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lin S., Kowalski D. Functional equivalency and diversity of cis-acting elements among yeast replication origins. Mol Cell Biol. 1997 Sep;17(9):5473–5484. doi: 10.1128/mcb.17.9.5473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lustig A. J. Mechanisms of silencing in Saccharomyces cerevisiae. Curr Opin Genet Dev. 1998 Apr;8(2):233–239. doi: 10.1016/s0959-437x(98)80146-9. [DOI] [PubMed] [Google Scholar]
  20. Mahoney D. J., Broach J. R. The HML mating-type cassette of Saccharomyces cerevisiae is regulated by two separate but functionally equivalent silencers. Mol Cell Biol. 1989 Nov;9(11):4621–4630. doi: 10.1128/mcb.9.11.4621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mahoney D. J., Marquardt R., Shei G. J., Rose A. B., Broach J. R. Mutations in the HML E silencer of Saccharomyces cerevisiae yield metastable inheritance of transcriptional repression. Genes Dev. 1991 Apr;5(4):605–615. doi: 10.1101/gad.5.4.605. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Newlon C. S., Collins I., Dershowitz A., Deshpande A. M., Greenfeder S. A., Ong L. Y., Theis J. F. Analysis of replication origin function on chromosome III of Saccharomyces cerevisiae. Cold Spring Harb Symp Quant Biol. 1993;58:415–423. doi: 10.1101/sqb.1993.058.01.048. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Palacios DeBeer M. A., Fox C. A. A role for a replicator dominance mechanism in silencing. EMBO J. 1999 Jul 1;18(13):3808–3819. doi: 10.1093/emboj/18.13.3808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Paulovich A. G., Margulies R. U., Garvik B. M., Hartwell L. H. RAD9, RAD17, and RAD24 are required for S phase regulation in Saccharomyces cerevisiae in response to DNA damage. Genetics. 1997 Jan;145(1):45–62. doi: 10.1093/genetics/145.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Rao H., Stillman B. The origin recognition complex interacts with a bipartite DNA binding site within yeast replicators. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):2224–2228. doi: 10.1073/pnas.92.6.2224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rashid M. B., Shirahige K., Ogasawara N., Yoshikawa H. Anatomy of the stimulative sequences flanking the ARS consensus sequence of chromosome VI in Saccharomyces cerevisiae. Gene. 1994 Dec 15;150(2):213–220. doi: 10.1016/0378-1119(94)90429-4. [DOI] [PubMed] [Google Scholar]
  30. Reynolds A. E., McCarroll R. M., Newlon C. S., Fangman W. L. Time of replication of ARS elements along yeast chromosome III. Mol Cell Biol. 1989 Oct;9(10):4488–4494. doi: 10.1128/mcb.9.10.4488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rivier D. H., Ekena J. L., Rine J. HMR-I is an origin of replication and a silencer in Saccharomyces cerevisiae. Genetics. 1999 Feb;151(2):521–529. doi: 10.1093/genetics/151.2.521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Rowley A., Cocker J. H., Harwood J., Diffley J. F. Initiation complex assembly at budding yeast replication origins begins with the recognition of a bipartite sequence by limiting amounts of the initiator, ORC. EMBO J. 1995 Jun 1;14(11):2631–2641. doi: 10.1002/j.1460-2075.1995.tb07261.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Santocanale C., Diffley J. F. A Mec1- and Rad53-dependent checkpoint controls late-firing origins of DNA replication. Nature. 1998 Oct 8;395(6702):615–618. doi: 10.1038/27001. [DOI] [PubMed] [Google Scholar]
  35. Santocanale C., Diffley J. F. ORC- and Cdc6-dependent complexes at active and inactive chromosomal replication origins in Saccharomyces cerevisiae. EMBO J. 1996 Dec 2;15(23):6671–6679. [PMC free article] [PubMed] [Google Scholar]
  36. Santocanale C., Sharma K., Diffley J. F. Activation of dormant origins of DNA replication in budding yeast. Genes Dev. 1999 Sep 15;13(18):2360–2364. doi: 10.1101/gad.13.18.2360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Scherer S., Davis R. W. Replacement of chromosome segments with altered DNA sequences constructed in vitro. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4951–4955. doi: 10.1073/pnas.76.10.4951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Shirahige K., Hori Y., Shiraishi K., Yamashita M., Takahashi K., Obuse C., Tsurimoto T., Yoshikawa H. Regulation of DNA-replication origins during cell-cycle progression. Nature. 1998 Oct 8;395(6702):618–621. doi: 10.1038/27007. [DOI] [PubMed] [Google Scholar]
  39. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Stone E. M., Pillus L. Silent chromatin in yeast: an orchestrated medley featuring Sir3p [corrected]. Bioessays. 1998 Jan;20(1):30–40. doi: 10.1002/(SICI)1521-1878(199801)20:1<30::AID-BIES6>3.0.CO;2-W. [DOI] [PubMed] [Google Scholar]
  41. Theis J. F., Newlon C. S. The ARS309 chromosomal replicator of Saccharomyces cerevisiae depends on an exceptional ARS consensus sequence. Proc Natl Acad Sci U S A. 1997 Sep 30;94(20):10786–10791. doi: 10.1073/pnas.94.20.10786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Vujcic M., Miller C. A., Kowalski D. Activation of silent replication origins at autonomously replicating sequence elements near the HML locus in budding yeast. Mol Cell Biol. 1999 Sep;19(9):6098–6109. doi: 10.1128/mcb.19.9.6098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Walker S. S., Francesconi S. C., Eisenberg S. A DNA replication enhancer in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4665–4669. doi: 10.1073/pnas.87.12.4665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Weinberger M., Trabold P. A., Lu M., Sharma K., Huberman J. A., Burhans W. C. Induction by adozelesin and hydroxyurea of origin recognition complex-dependent DNA damage and DNA replication checkpoints in Saccharomyces cerevisiae. J Biol Chem. 1999 Dec 10;274(50):35975–35984. doi: 10.1074/jbc.274.50.35975. [DOI] [PubMed] [Google Scholar]
  45. Zhu J., Newlon C. S., Huberman J. A. Localization of a DNA replication origin and termination zone on chromosome III of Saccharomyces cerevisiae. Mol Cell Biol. 1992 Oct;12(10):4733–4741. doi: 10.1128/mcb.12.10.4733. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES