REPORTS ecules was FLP-mediated recombination. FLP recombinase catalyzes the entire recom- bination reaction, requiring no other proteins (26) and leaving no substrates for repair syn- thesis. Also, no repair-coupled synthesis of the excised HMR molecules was detected by transient resistance to Dpn I cleavage. Thus, it was unlikely that replication-coupled pro- cess contributed to the quantitative silencing observed here. We are unaware of any evi- dence that mutations or damage occurs more readily on circular plasmids in yeast than on the chromosome. In addition to offering a new mechanistic insight on silencing, these data place renewed importance on the role of proteins involved in DNA replication, such as PCNA, Rfclp, Asflp, Dna2p, and CAF-1, in silencing. An important challenge is to learn how those proteins affect silencing when silencing can be mechanistically divorced from both repli- cation initiation and from the passage of a replication fork. PCNA left behind on a pre- viously replicated template can mark that template as "competent" for CAF-l-depen- dent chromatin assembly (33). If PCNA from the previous cell cycle remains associated with HMR upon entering the subsequent G, phase, it may be excised with HMR and therefore available to establish heterochroma- tin. The efficiency of silencing observed here would require that some feature of HMR causes retention of PCNA. Alternatively, these proteins may have a role in silencing other than in its establishment (18). For ex- ample, once heterochromatin is established at a locus, it must be maintained throughout that cell cycle and duplicated in each subsequent cell cycle. Indeed, recent data underscore the dynamic nature of heterochromatin composi- tion in vivo, even on nonreplicating DNA molecules (3). Perhaps proteins like CAFl and PCNA have a replication-coupled role in the inheritance of heterochromatin at HMR, or possibly in its maintenance. The results of this study have reframed the essential outstanding issues in establishing heterochromatin. One goal now is to learn what replication-independent event happens in this cell cycle window that is essential for silencing. The second goal is to uncover how replication proteins play a role in silencing when replication itself is not required. Both questions should provide fundamental in- sights into how cells assemble specific struc- tures of chromatin in a spatially and tempo- rally organized manner. References and Notes 1. J. Z. Dalgaard, A. j. Klar, Nature 400, 181 (1999). 2. X. Bi, j. R. Broach, Mol. Cell. Biol. 17, 7077 (1997). 3. T.-H. Cheng, M. R. Gartenberg. Genes Dev. 14, 452 (2000). 4. A. Schmid, K.-D. Fascher, W. Horz, Cell 71, 853 (1992). 5. 5. Loo, J. Rine, Ann. Rev. Cell Dev. Biol. 11, 519 (1995). 6. A. Hecht, T. Laroche, 5. Strahl-Bolsinger, 5. M. Gasser, 24. C. A. Fox, S. Loo, A. Dillin, j. Rine, Genes Dev. 9, 91 1 M. Grunstein, Cell 80, 583 (1995). (1995). 7. S. Imai, C. M. Armstrong, M. Kaeberlein, L. Guarente, 25. j. R. Broach, F. C. Volkert, in The Molecular and Nature 403, 795 (2000). Cellular Biology of the Yeast Saccharomyces: Genome 8. K. A. Gardner, j. Rine, C. A. Fox, Genetics 151, 31 Dynamics, Protein Synthesis and Energetics, j. R. (1999). Broach, j. R. Pringle, E. W. Jones, Eds. (Cold Spring 9. T. Triolo, R. Sternglanz, Nature 381, 251 (1996). Harbor Laboratory Press, Plainview, NY, 1991), vol I. 10. A. M. Miller, K. A. Nasmyth, Nature 312, 247 (1984). pp. 297-331. 11. D. H. Rivier, j. L. Ekena, j. Rine, Genetics 151, 521 26. L. Meyer-Leon,C. A. Gates, j. M. Attwood, E. A. Wood. (1999). M. M. Cox, Nucleic Acids Res. 15. 6469 (1987). 27. A. L. Kirchmaier, J. Rine, data not shown. 12. M. Foss, F. j. McNally, P. Laurenson, J. Rine, Science 28. Y.-C. Li, T. H. Cheng, M. R. Gartenberg, Science 291. 262, 1838 (1993). 650 (2001). 13. 5. Loo et al., Mol. Biol. Cell 6, 741 (1995). 29. S. G. Holmes, j. R. Broach, Genes Dev. 10, 1021 14. G. Micklem, A. Rowley, J. Harwood, K. Nasmyth, (1996). j. F. X. Diffley, Nature 366, 87 (1993). 30. Web fig. 1, Web table 1, and supplemental text are 15, j. 5. Smith, E. Caputo, j. D. Boeke, Mol. Cell. Biol. 19, available at Science Online at www.sciencemag.org/ 3184 (1999). cgi/content/full/291/5504/646/DCl. 16. M. S. Singer et al., Genetics 150, 613 (1998). 31. K. L. Friedman, M. K. Raghuraman, W. L. Fangman, B. j. 17. P. D. Kaufman, R. Kobayashi, B. Stillrnan, Genes Dev. Brewer, J. Cell Sci. Suppl. 19, 51 (1995). 11, 345 (1997). 32. j. Abraham, K. A. Nasmyth, J. N. Strathern, A. j. S. Klar, 18. S. Enomoto, 1. Berman, Genes Dev. 12, 219 (1998). j. B. Hicks, J. Mol. Biol. 176, 307 (1984). 19. P. Kaufman, personal communication. 33. K.-i. Shibahara, B. Stillrnan, Cell 96, 575 (1999). 20. Z. Zhang, K. Shibahara, B. Stillman, Nature 408, 221 34. We thank J. Broach for pFVl7: J. Gin and L. Rusche for (2000). technical assistance; P. Garber, L. Rusche, and P. 21. C. A. Fox, A. E. Ehrenhofer-Murray, S. Loo, J. Rine. Kaufman for helpful discussions; and L. Rusche. P. Science 276, 1547 (1997). Kaufman, M. Botchan, and B. Meyer for critical re- 22. S. P. Bell, J. Mitchell, J. Leber, R. Kobayashi. B. Stillrnan, view. Supported by NIHF32GM19392 (A.L.K.) and Cell 83, 563 (1995). NIHGM31105 (J.R.). . . 23. A. Dillin, j. Rine. Genetics 147, 1053 (1997). 14 August 2000: accepted 19 December 2000 Establishment of Transcriptional Silencing in the Absence of DNA Replication Yao-Cheng Li, Tzu-Hao Cheng,* Marc R. Cartenbergtl Transcriptional repression of the silent mating-type loci in Saccharomyces cerevisiae requires a cell cycle-dependent establishment step that is commonly assumed to involve DNA replication. Using site-specific recombination, we created a nonreplicating DNA ring in vivo to test directly the role of replication in establishment of silencing. Sirl was tethered to the ring following excision from the chromosome to activate a dormant silencer. We show here that silencing can be established in DNA that does not replicate. The silenced ring adopted structural features characteristic of bona fide silent chromatin, in- cluding an altered level of DNA supercoiling and reduced histone acetylation. In addition, the process required silencing factors Sir2, Sir3, and Sir4 and progression between early S and M phases of the cell cycle. The results indicate that passage of a replication fork is not the cell-cycle event required for establishment of silencing in yeast. Silencing of large chromosomal domains in- HML is governed by cis-acting sequences, volves specialized, heritable chromatin struc- known as the E and I silencers, which flank tures that repress transcription in a gene-inde- both loci and consist of various combinations of pendent fashion. The silent HM mating-type sites for Raplp, Abflp, and the multisubunit loci of budding yeast (HMR and HML) repre- origin recognition complex (ORC). Despite an sent well-studied examples of this type of tran- essential role for ORC in initiation of DNA scriptional control (I). Silencing of HMR and replication, substantial evidence indicates that this is not its function at silencers. Only a subset Department of Pharmacology,University of Medicine of silencers act as chromosomal replication or- and Dentistrv of New Iersev-Robert wood johnson igins and orc mutants have been isolated that Medical ~chiol, ~iscataway,-~~ are defective in replication initiation but not 08854, USA. - *Present address: Department of Genetics, Stanford ~ilencing (2. 3). Instead, the function of ORC at University School of Medicine, Stanford, CA 94305, silencers appears to be recruitment of Sirl (4 - USA. 6), which. along with the other silencer binding TMember, The Cancer Institute of New Jersey, New proteins. facilitates incorporation of Sir2, Sir3, Brunswick. N 1 08901. USA. whom should be addressed, E. and Sir4 into a heterochromatin-like structure, mail: gartenbe@umdnj.edu termed silent chromatin. Indeed. artificially 650 26 JANUARY 2001 VOL 291 SCIENCE www.sciencemag.org