Replication and segregation of plasmids containing cis-acting regulatory sites of silent mating-type genes in Saccharomyces cerevisiae are controlled by the SIR genes.

Abstract

In Saccharomyces cerevisiae, two cis-acting regulatory sites called E and I flank the silent mating-type gene, HMRa, and mediate SIR-dependent transcriptional repression of the al-a2 promoters. It has been shown previously that the E and I sites have plasmid replicator (ARS) activity. We show in this report that the ARS activity of the E and I sites is governed by the SIR genotype of the cell. In wild-type cells, a plasmid carrying the E site from HMRa (HMR E) in the vector YIp5 exhibited very high mitotic stability at a copy number of approximately 25 per cell. However, in sir2, sir3, or sir4 mutants, plasmids with HMR E had the low mitotic stability characteristic of plasmids containing ARSI, a SIR-independent replicator. Elevated mitotic stability of plasmids that carry HMR E is due to a segregation mechanism provided by SIR and HMR E. In sir2 and sir4 mutants, the plasmid copy number was significantly lowered, suggesting that these gene products also participate in the replication of plasmids carrying HMR E. The phenotype of point mutations introduced at an 11-base-pair ARS consensus sequence present at HMR E indicated that this sequence is functional but not absolutely required for autonomous replication of the plasmid and that it is not required for SIR-dependent mitotic stabilization. A plasmid carrying both a centromere and HMR E exhibited reduced mitotic stability in wild-type cells. This destabilization appeared to be due to antagonism between the segregation functions provided by the centromere and by HMR E.The genes HML and HMR encode cryptic copies of the MAT genes. HML, located on the left arm of chromosome III, encodes an unexpressed copy of the MATa genes (HMLo). HMR, located distal to MAT on the right arm of chromosome III, contains an unexpressed copy of the MATa genes (3,23,44,59). These duplicated genes serve as donor templates in HO-mediated mating-type interconversion (reviewed in reference 24). The expression of the genes at HML and HMR is repressed by the products of SIR], SIR2 (MARI), SIR3 (CMT), and SIR4 (21,33,35,47,48). Both HML and HMR are flanked by cis-acting sites, called E and I, that are also required for transcriptional repression. Deletion and insertion analyses indicate that the E site is contained within a 220-base-pair (bp) region and the I site within an 85-bp region at HMR (1, 19). Presumably, E and I are the sites through which the SIR proteins mediate transcriptional repression of HML and HMR. Indeed, DNA sequence analysis of the E and I elements has revealed several short conserved blocks of homology, some of which may be sites of SIR action (19).In addition to their role in SIR-mediated transcriptional repression, the E and I elements allow plasmids to replicate autonomously in yeasts (1,10,19 chance and may represent a mechanistic link between the processes of DNA replication and transcriptional repression. In this regard, the onset of SIR-mediated repression at HMRa requires transit through the S phase of the cell cycle (42). It is not known whether the requirement ...

Silent domains are assembled continuously from the telomere and are defined by promoter distance and strength, and by SIR3 dosage.

“…All other plasmids used for strain construction have been described previously (Ivy et al 1986;Kimmerly and Rine 1987;Gottschling et al 1990;Aparicio et al 1991 ).…”

Conditional silencing: the HMRE mating-type silencer exerts a rapidly reversible position effect on the yeast HSP82 heat shock gene.

“…This step was taken to minimize the SIR-dependent centromeric and ARS activities of the HMR silencers, E and I (9,34), in an attempt to optimize the probability of chromosomal integration. We found that gene transplacement was greatly facilitated in a sir4 background; in SIR' recipient cells, uracil prototrophs invariably harbored the HMR/ HSP82 fragments as multicopy episomes.…”

A DNA polymerase mutation that suppresses the segregation bias of an ARS plasmid in Saccharomyces cerevisiae.

“…One class of mutations affect segregation when the plasmid contains the HMRE ARS from the silencer of mating type information on the right arm of chromosome III (13). In wild-type strains, plasmids with the HMRE ARS are comparatively stable because they segregate randomly.…”