title: Variants of the Sir4 Coiled-Coil Domain Improve Binding to Sir3 for Heterochromatin Formation inSaccharomyces cerevisiae.
abstract: Heterochromatin formation in the yeastSaccharomyces cerevisiaeis characterized by the assembly of the Silent Information Regulator (SIR) complex, which consists of the histone deacetylase Sir2 and the structural components Sir3 and Sir4, and binds to unmodified nucleosomes to provide gene silencing. Sir3 contains an AAA+ATPase-like domain, and mutations in an exposed loop on the surface of this domain abrogate Sir3 silencing functionin vivo, as wellin vitrobinding to the Sir2/Sir4 subcomplex. Here, we found that the removal of a single methyl group in the C-terminal coiled-coil domain (mutation T1314S) of Sir4 was sufficient to restore silencing at the silent mating-type lociHMRandHMLto a Sir3 version with a mutation in this loop. Restoration of telomeric silencing required further mutations of Sir4 (E1310V and K1325R). Significantly, these mutations in Sir4 restoredin vitrocomplex formation between Sir3 and the Sir4 coiled-coil, indicating that the improved affinity between Sir3 and Sir4 is responsible for the restoration of silencing. Altogether, these observations highlight remarkable properties of selected amino-acid changes at the Sir3-Sir4 interface that modulate the affinity of the two proteins.
authors.affiliation: Institut für Biologie, Humboldt-Universität zu Berlin, Berlin 10099, Germany.
authors.forename: Anke
authors.initials: A
authors.lastname: Samel
authors.affiliation: Department of Biochemistry, Microbiology and Immunology, Ottawa Institute of Systems Biology, University of Ottawa, Ontario ON K1H 8M5, Canada.
authors.forename: Adam
authors.initials: A
authors.lastname: Rudner
authors.affiliation: Institut für Biologie, Humboldt-Universität zu Berlin, Berlin 10099, Germany ann.ehrenhofer-murray@hu-berlin.de.
authors.forename: Ann E
authors.identifier: 0000-0001-8709-1942
authors.initials: AE
authors.lastname: Ehrenhofer-Murray
mesh_terms: D020868:Gene Silencing; D056426:Genetic Loci; D006570:Heterochromatin / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D000072417:Protein Domains; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013489:Suppression, Genetic; D016615:Telomere / Q000378:metabolism
chemical_list: D006570:Heterochromatin; D050505:Mutant Proteins; C084122:SIR3 protein, S cerevisiae; C061573:SIR4 protein, S cerevisiae; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae
keywords: Sir1; chromatin; gene silencing; repression