Establishment of transcriptional silencing in the absence of DNA replication

Transcriptional repression of the silent mating-type Loci in Saccharomyces cerevisiae requires a cell cycle-dependent establishment step that is commo nly assumed to involve DNA replication. Using site-specific recombination, we created a nonreplicating DNA ring in vivo to test directly the role of r eplication in establishment of silencing. Sir1 was tethered to the ring fol lowing 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 sile nt chromatin, including an altered Level of DNA supercoiling and reduced hi stone acetylation. In addition, the process required silencing factors Sir2 , Sir3, and Sir4 and progression between early S and M phases of the cell c ycle. The results indicate that passage of a replication fork is not the ce ll-cycle event required for establishment of silencing in yeast.