Kluyveromyces lactis Sir2p regulates cation sensitivity and maintains a specialized chromatin structure at the cryptic alpha-locus.

Abstract

In Saccharomyces cerevisiae, transcriptional silencing of the cryptic mating type loci requires the formation of a heterochromatin-like structure, which is dependent on silent information regulator (Sir) proteins and DNA sequences, called silencers. To learn more about silencing, we characterized the mating type loci from the yeast Kluyveromyces lactis. The K. lactis MAT, HMRa, and HMLalpha loci shared flanking DNA sequences on both sides of the loci presumably acting as recombinational targets during mating type switching. HMRa contained two genes, the a1 gene similar to the Saccharomyces a1 gene and the a2 gene similar to mating type genes from other yeasts. K. lactis HMLalpha contained three genes, the alpha1 and alpha2 genes, which were similar to their Saccharomyces counterparts, and a novel third gene, alpha3. A dam-methylase assay showed Sir-dependent, but transcription-independent changes of the chromatin structure of the HMLalpha locus. The HMLalpha3 gene did not appear to be part of the silent domain because alpha3p was expressed from both MATalpha3 and HMLalpha3 and sir mutations failed to change the chromatin structure of the HMLalpha3 gene. Furthermore, a 102-bp silencer element was isolated from the HMLalpha flanking DNA. HMLalpha was also flanked by an autonomously replicating sequence (ARS) activity, but the ARS activity did not appear to be required for silencer function. K. lactis sir2 strains grown in the presence of ethidium bromide (EtBr) accumulated the drug, which interfered with the essential mitochondrial genome. Mutations that bypassed the requirement for the mitochondrial genome also bypassed the EtBr sensitivity of sir2 strains. Sir2p localized to the nucleus, indicating that the role of Sir2p to hinder EtBr accumulation was an indirect regulatory effect. Sir2p was also required for growth in the presence of high concentrations of Ni(2+) and Cu(2+).