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

In Saccharomyces cerevisiae, transcriptional silencing of the cryptic matin g type loci requires the formation of a heterochromatin-like structure, whi ch is dependent on silent information regulator (Sir) proteins and DNA sequ ences, called silencers. To learn more about silencing, we characterized th e mating type loci from the yeast Kluyveromyces lactis. The K. lactis MAT, HMRa, and HML alpha loci shared flanking DNA sequences on both sides of the loci presumably acting as recombinational targets during mating type switc hing. HMR alpha contained two genes, the a1 gene similar to the Saccharomyc es a1 gene and the a2 gene similar to mating type genes from other yeasts. K. lactis HML alpha contained three genes, the alpha 1 and alpha 2 genes, w hich were similar to their Saccharomyces counterparts, and a novel third ge ne, alpha 3. A dam-methylase assay showed Sir-dependent, but transcription- independent changes of the chromatin structure of the HML alpha locus. The HML alpha 3 gene did not appear to be part of the silent domain because alp ha 3p was expressed from both MAT alpha 3 and HML alpha 3 and sir mutations failed to change the chromatin structure of the HML alpha 3 gene. Furtherm ore, a 102-bp silencer element was isolated from the HML alpha flanking DNA . HML alpha was also flanked by an autonomously replicating sequence (ART) activity, but the ARS activity did not appear to be required for silencer f unction. K. lactis sir2 strains grown in the presence of ethidium bromide ( EtBr) accumulated the drug, which interfered with the essential mitochondri al genome. Mutations that bypassed the requirement for the mitochondrial ge nome 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 t he presence of high concentrations of Ni2+ and Cu2+.