S phase and establishment of silencing in Saccharomyces
cerevisiae .
Ann L. Kirchmaier (1), Jasper Rine
(2)
(1) Department of Biochemistry, Purdue University, 1153
Biochemistry, West Lafayette, IN 47907, USA; (2) Department of
Molecular and Cell Biology, 401 Barker Hall, University of California,
Berkeley, CA 94709 USA
In Saccharomyces cerevisiae, silencing
the mating-type loci, HML and HMR, by heterochromatin
requires a cell cycle event that occurs in S phase. We found that this
cell cycle-restricted event is neither the initiation of replication at
the silencers nor the passage of the DNA replication fork through
HMR. Previous results of others implied that the critical S
phase event in silencing occurs after the hydroxyurea (HU) block in mid-S phase. However, we have shown that an early S phase event is
sufficient for silencing and that HU treatment itself blocks silencing
on replication-competent chromosomal templates but not on templates that
cannot be replicated. The effects of HU treatment on heterochromatin
formation are MEC1-independent. We have identified discrete,
separable stages in heterochromatin formation. These are the loading of
Sir1 protein and then the other Sir proteins on the silencer and the
spreading of the Sir proteins throughout the silencer regions. Spreading
requires the deacetylase activity of Sir2p. Remarkably, the S phase
requirement for silencing appears to act post-recruitment and spreading
of Sir proteins and after deacetylation of histones. Paradoxically,
mutations of several proteins involved in DNA replication, including
PCNA, CAF-1 and Asf1p affect silencing despite a lack of a requirement
for replication in silencing. We have shown that PCNA is required for
silencing of loci that are not replicated during the cell cycle in which
they are silenced.
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