A Suppressor of Mating-Type Locus Mutations in SACCHAROMYCES CEREVISIAE: Evidence for and Identification of Cryptic Mating-Type Loci

A mutation has been identified that suppresses the mating and sporulation defects of all mutations in the mating-type loci of S. cerevisiae. This suppressor, sir1-1, restores mating ability to mat alpha 1 and mat alpha 2 mutants and restores sporulation ability to mat alpha 2 and mata1 mutants. MATa sir1-1 strains exhibit a polar budding pattern and have reduced sensitivity to alpha-factor, both properties of a/alpha diploids. Furthermore, sir1-1 allows MATa/MATa, mat alpha 1/mat alpha/, and MAT alpha/MAT alpha strains to sporulate efficiently. All actions of sir1-1 are recessive to SIR1. The ability of sir1-1 to supply all functions necessary for mating and sporulation and its effects in a cells are explained by proposing that sir1-1 allows expression of mating type loci which are ordinarily not expressed. The ability of sir1-1 to suppress the mat alpha 1-5 mutation is dependent on the HMa gene, previously identified as required for switching of mating types from a to alpha. Thus, as predicted by the cassette model, HMa is functionally equivalent to MAT alpha since it supplies functions of MAT alpha. We propose that sir1-1 is defective in a function. Sir ("Silent-information regulator"), whose role may be to regulate expression of HMa and HM alpha.     

The dosage of chromatin proteins affects transcriptional silencing and DNA repair in Saccharomyces cerevisiae

Alterations in protein composition or dosage within chromatin may trigger changes in processes such as gene expression and DNA repair. Through transposon mutagenesis and targeted gene deletions in haploids and diploids of Saccharomyces cerevisiae, we identified mutations that affect telomeric silencing in genes encoding telomere-associated Sir4p and Yku80p and chromatin remodeling ATPases Ies2p and Rsc1p. We found that sir4/SIR4 heterozygous diploids efficiently silence the mating type locus HMR but not telomeres, and diploids heterozygous for yku80 and ies2 mutations are inefficient at DNA repair. In contrast, strains heterozygous for most chromatin remodeling ATPase mutations retain wild-type silencing and DNA repair levels. Thus, in diploids, chromatin structures required for DNA repair and telomeric silencing are sensitive to dosage changes.     

Locus specificity determinants in the multifunctional yeast silencing protein Sir2

Yeast SIR2, the founding member of a conserved gene family, acts to modulate chromatin structure in three different contexts: silent (HM) mating-type loci, telomeres and rDNA. At HM loci and telomeres, Sir2p forms 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. A surprising number of these mutations are dominant. Several rDNA silencing mutants display a Sir2p nucleolar localization defect that correlates with reduced Net1p binding. Although the molecular defect in HM/telomere-specific mutants is unclear, they mimic an age-related phenotype where Sir3p and Sir4p relocalize to the nucleolus. Artificial targeting can circumvent the silencing defect in a subset of mutants from both classes. These results define distinct functional domains of Sir2p and provide evidence for additional Sir2p-interacting factors with locus-specific silencing functions.     

The clustering of telomeres and colocalization with Rap1, Sir3, and Sir4 proteins in wild-type Saccharomyces cerevisiae

We have developed a novel technique for combined immunofluorescence/in situ hybridization on fixed budding yeast cells that maintains the three-dimensional structure of the nucleus as monitored by focal sections of cells labeled with fluorescent probes and by staining with a nuclear pore antibody. Within the resolution of these immunodetection techniques, we show that proteins encoded by the SIR3, SIR4, and RAP1 genes colocalize in a statistically significant manner with Y' telomere- associated DNA sequences. In wild-type cells the Y' in situ hybridization signals can be resolved by light microscopy into fewer than ten foci per diploid nucleus. This suggests that telomeres are clustered in vegetatively growing cells, and that proteins essential for telomeric silencing are concentrated at their sites of action, i.e., at telomeres and/or subtelomeric regions. As observed for Rap1, the Sir4p staining is diffuse in a sir3- strain, and similarly, Sir3p staining is no longer punctate in a sir4- strain, although the derivatized Y' probe continues to label discrete sites in these strains. Nonetheless, the Y' FISH is altered in a qualitative manner in sir3 and sir4 mutant strains, consistent with the previously reported phenotypes of shortened telomeric repeats and loss of telomeric silencing.     

SIR Functions Are Required for the Toleration of an Unrepaired Double-Strand Break in a Dispensable Yeast Chromosome

Unrepaired DNA double-strand breaks (DSBs) typically result in G(2) arrest. Cell cycle progression can resume following repair of the DSBs or through adaptation to the checkpoint, even if the damage remains unrepaired. We developed a screen for factors in the yeast Saccharomyces cerevisiae that affect checkpoint control and/or viability in response to a single, unrepairable DSB that is induced by HO endonuclease in a dispensable yeast artificial chromosome containing human DNA. SIR2, -3, or -4 mutants exhibit a prolonged, RAD9-dependent G(2) arrest in response to the unrepairable DSB followed by a slow adaptation to the persistent break, leading to division and rearrest in the next G(2). There are a small number of additional cycles before permanent arrest as microcolonies. Thus, SIR genes, which repress silent mating type gene expression, are required for the adaptation and the prevention of indirect lethality resulting from an unrepairable DSB in nonessential DNA. Rapid adaptation to the G(2) checkpoint and high viability were restored in sir(-) strains containing additional deletions of the silent mating type loci HML and HMR, suggesting that genes under mating type control can reduce the toleration of a single DSB. However, coexpression of MATa1 and MATalpha2 in Sir(+) haploid cells did not lead to lethality from the HO-induced DSB, suggesting that toleration of an unrepaired DSB requires more than one Sir(+) function.