Abstract

We have examined the re-establishment of HIMRE mediated silencing function on the transcriptional activity of yeast heat shock gene HSP82. To test whether the onset of SIR repression can occur in growing cells in the presence of a potent inhibitor of DNA replication, HMRa/HSP82 strains with SIR4^- and SIR4S^+ genetic backgrounds were arrested in S phase by incubation of a culture in 200 mM hydroxyurea for 120 min. It was clear that following a 20 minute heat shock, silencing of the HMRa/HSP82 allele in cells pretreated with hydroxyurea does occur in a SIR4-dependen fashion, even though the kinetics of repression appears to be substantially delayed. We also have tested whether re- establishment of silencing at the HMR/hsp82 locus can occur in G1-arrested cells. Cell cycle arrest at G1 phase was achieved by treatment of early log a cell cultures with α-factor mating pheromone, which induces G1 arrest. The result suggests that passage through S phase (and therefore DNA replication) is nor required for re-establishing silencer-mediated repression at the HMNRa/HSP82 locus. Finally, to test whether de novo protein synthesis is required for re-establishment of silencer-mediated repression, cells were pretreated with cycloheximide (500 ㎍/㎖) 120 min. It was apparent that inhibiting protein synthesis delays, but does not prevent, re-establishment of silencer-mediated repression. Altogether, these results indicate that re-establishment of silencer-mediated repression is not dependent on the DNA replication and has no requirement for protein synthesis.

• Keywords: DNA replication; saccharomyces cerevisiae; silencer