SIR2, the founding member of a conserved gene
family, acts to modulate chromatin structure at silent (HM)
mating-type loci, telomeres and rDNA. At HM loci and telomeres
Sir2p works in a complex with Sir3p and Sir4p. However, Sir2p's role
in rDNA silencing is SIR3/4-independent, requiring instead an
essential nucleolar protein, Net1p. We describe two novel classes of
SIR2 mutations specific to either HM/telomere or rDNA
silencing. Despite their opposite effects, both classes of mutations
cluster in the same two regions of Sir2p, each of which borders on a
conserved core domain. Although we screened for (specific) loss of
function, a large number of the mutations we isolated are dominant to
SIR2. Several rDNA silencing mutants display a Sir2p nucleolar
localization defect that correlates with reduced Net1p
binding. Interestingly, a subset of these mutants are also defective
in a SIR2-dependent meiotic checkpoint. Unexpectedly, none of
the HM/telomere-specific mutations appear to affect the
Sir2p-Sir4p interaction. Instead, these mutants mimic an age-related
phenotype where Sir3p and Sir4p relocalize to the nucleolus,
presumably in association with the mutant Sir2 protein. This might be
the result of a loss of interaction between Sir2p and another factor,
which occurs normally in old wild-type cells. Taken together, these
results define distinct functional domains of Sir2p and provide
evidence for additional Sir2p-interacting factors with locus-specific
silencing functions.
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