The Role of Sas2, an Acetyltransferase Homologue of Saccharomyces Cerevisiae, in Silencing and Orc Function

Silencing at the cryptic mating-type loci HML and HMR of Saccharomyces cerevisiae requires regulatory sites called silencers. Mutations in the Rap1 and Abf1 binding sites of the HMR-E silencer (HMRa-e**) cause the silencer to be nonfunctional, and hence, cause derepression of HMR. Here, we have isolated and characterized mutations in SAS2 as second-site suppressors of the silencing defect of HMRa-e**. Silencing conferred by the removal of SAS2 (sas2Δ) depended upon the integrity of the ARS consensus sequence of the HMR-E silencer, thus arguing for an involvement of the origin recognition complex (ORC). Restoration of silencing by sas2Δ required ORC2 and ORC5, but not SIR1 or RAP1. Furthermore, sas2Δ suppressed the temperature sensitivity, but not the silencing defect of orc2-1 and orc5-1. Moreover, sas2Δ had opposing effects on silencing of HML and HMR. The putative Sas2 protein bears similarities to known protein acetyltransferases. Several models for the role of Sas2 in silencing are discussed.     

Analysis of chromosome III replicators reveals an unusual structure for the ARS318 silencer origin and a conserved WTW sequence within the origin recognition complex binding site

Saccharomyces cerevisiae chromosome III encodes 11 autonomously replicating sequence (ARS) elements that function as chromosomal replicators. The essential 11-bp ARS consensus sequence (ACS) that binds the origin recognition complex (ORC) has been experimentally defined for most of these replicators but not for ARS318 (HMR-I), which is one of the HMR silencers. In this study, we performed a comprehensive linker scan analysis of ARS318. Unexpectedly, this replicator depends on a 9/11-bp match to the ACS that positions the ORC binding site only 6 bp away from an Abf1p binding site. Although a largely inactive replicator on the chromosome, ARS318 becomes active if the nearby HMR-E silencer is deleted. We also performed a multiple sequence alignment of confirmed replicators on chromosomes III, VI, and VII. This analysis revealed a highly conserved WTW motif 17 to 19 bp from the ACS that is functionally important and is apparent in the 228 phylogenetically conserved ARS elements among the six sensu stricto Saccharomyces species.     

Purification and characterization of proteins that bind to yeast ARSs

Two proteins that bind to yeast ARS DNA have been purified using conventional and oligonucleotide affinity chromatography. One protein has been purified to homogeneity and has a mass of 135 kDa. Competitive binding studies and DNase I footprinting show that the protein binds to a sequence about 80 base pairs away from the core consensus in the region known as domain B. This region has previously been shown to be required for efficient replication of plasmids carrying ARS1 elements. To investigate further whether the protein might have a function related to the ability of ARSs to act as replicators, binding to another ARS was tested. The protein binds to the functional ARS adjacent to the silent mating type locus HMR, called the HMR-E ARS, about 60 base pairs from the core consensus sequence. Surprisingly, there is little homology between the binding site at the HMR-E ARS and the binding site at ARS1. The 135-kDa protein is probably the same as ABF-I (SBF I) (Shore, D., Stillman, D. J. Brand, A. H., and Nasmyth, K. A. (1987) EMBO J. 6, 461-467; Buchman, A. R., Kimmerly, W. J., Rine, J., and Kornberg, R. D. (1988) Mol. Cell. Biol. 8, 210-225). A second DNA-binding protein was separated from ABF-I during later stages of the purification. This protein, which we designate ABF-III, also binds specifically to the ARS1 sequence, as shown by DNase I footprinting, at a site adjacent to the ABF-I recognition site. Purification of these two ARS binding proteins should aid in our understanding of the complex mechanisms that regulate eukaryotic DNA replication.     

A role for the Saccharomyces cerevisiae RENT complex protein Net1 in HMR silencing

Silencing in the yeast Saccharomyces cerevisiae is known in three classes of loci: in the silent mating-type loci HML and HMR, in subtelomeric regions, and in the highly repetitive rDNA locus, which resides in the nucleolus. rDNA silencing differs markedly from the other two classes of silencing in that it requires a DNA-associated protein complex termed RENT. The Net1 protein, a central component of RENT, is required for nucleolar integrity and the control of exit from mitosis. Another RENT component is the NAD(+)-dependent histone deacetylase Sir2, which is the only silencing factor known to be shared among the three classes of silencing. Here, we investigated the role of Net1 in HMR silencing. The mutation net1-1, as well as NET1 expression from a 2micro-plasmid, restored repression at silencing-defective HMR loci. Both effects were strictly dependent on the Sir proteins. We found overexpressed Net1 protein to be directly associated with the HMR-E silencer, suggesting that Net1 could interact with silencer binding proteins and recruit other silencing factors to the silencer. In agreement with this, Net1 provided ORC-dependent, Sir1-independent silencing when artificially tethered to the silencer. In contrast, our data suggested that net1-1 acted indirectly in HMR silencing by releasing Sir2 from the nucleolus, thus shifting the internal competition for Sir2 from the silenced loci toward HMR.