Regulation of replication initiation and HML silencing in Saccharomyces cerevisiae
The Saccharomyces cerevisiae origin recognition complex (ORC) displays a dual role in
silencing and initiation of DNA replication, but requires the contribution of auxiliary factors
such as Rap1 or Sum1 for full initiation capacity. In this study, the influence of ORC and
factors binding in the vicinity of ORC on origin activity and HML silencing was analysed.
The silent mating-type loci HML and HMR of S. cerevisiae contain mating-type information
that is permanently repressed. This silencing is mediated by flanking sequence elements, the
E- and I-silencers. They contain combinations of binding sites for Rap1, Abf1 and Sum1 as
well as for ORC. Together, they recruit other silencing factors, foremost the repressive
Sir2/Sir3/Sir4 complex, to establish heterochromatin-like structures at the HM loci. However,
the HM silencers exhibit considerable functional redundancy, which has hampered the
identification of further silencing factors. Therefore, a synthetic, minimal HML-E silencer
(HML-SS ΔI) that lacked this redundancy was constructed during the course of this study. It
consisted solely of Rap1 and ORC binding sites and the D2 element, a Sum1 binding site, and
all three elements were crucial for minimal HML silencing. This silencer was sensitive to a
mutation in RAP1, rap1-12, but less sensitive to orc mutations or sum1Δ. Moreover, deletions
of SIR1 and DOT1 led to complete derepression of the HML-SS ΔI silencer. This fully
functional, minimal HML-E silencer will therefore be useful to identify novel factors involved
in HML silencing.
Replication initiation at origins of replication in the yeast genome takes place on chromatin as
a template, raising the question how histone modifications, for instance histone acetylation,
influence origin firing. Initiation requires binding of ORC to a consensus sequence within
origins and of other proteins, for instance Sum1, to recognition sites in the vicinity of ORC to
support initiation. Sum1 is part of the Sum1/Rfm1/Hst1 complex that represses meiotic genes
during vegetative growth via histone deacetylation by the histone deacetylase (HDAC) Hst1.
In this study, it was found that Sum1 functioned in initiation as a component of this complex,
implying a role for histone deacetylation in origin activity. Several origins were identified in
the yeast genome whose activity depended on both Sum1 and Hst1. Importantly, sum1Δ or
hst1Δ caused a significant increase in histone H4 lysine 5 (H4 K5) acetylation levels, but not
other H4 acetylation sites, at those origins. Furthermore, mutation of lysines to glutamines in
the H4 tail, which imitates the constantly acetylated state, resulted in a reduction of origin
activity comparable to that in the absence of Hst1, showing that deacetylation of H4 was
important for full initiation capacity of these origins.
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