The role of the N-terminal acetyltransferase NatA in transcriptional silencing in Saccharomyces cerevisiae DISSERTATION

bei der Modellierung der Chromatinstruktur. Abstract Abstract N α-acetylation, one of the most abundant eukaryotic protein modifications, is catalyzed by N-terminal acetyltransferases (NATs). NatA, the major NAT in Saccharomyces cerevisiae, consists of the subunits Nat1, Ard1 and Nat5 and is necessary for the assembly of repressive chromatin structures at the silent mating type loci and telomeres. This thesis shows that NatA also acts in rDNA repression and it provides the first direct evidence for the functional regulation of the silencing factors Orc1 and Sir3 by NatA-dependent N α-acetylation. Orc1, the large subunit of the origin recognition complex (ORC), was N α-acetylated in vivo by NatA. Mutations that abrogated this acetylation caused strong telomeric derepression. NatA functioned genetically through the ORC binding site of the HMR-E silencer. Direct tethering of Orc1 to HMR-E circumvented the requirement for NatA in silencing. The synthetic lethality of nat1∆ orc2-1 double mutants further supported a functional link between NatA and ORC. Sir3 was also indentified as a NatA substrate. Its localization to perinuclear foci was NAT1 dependent. Unacetylated sir3 orc1 double mutants did not resemble the nat1∆ silencing phenotype. Thus, we suggest that further silencing components require NatA-dependent N α-acetylation for their function. We further identified the N-terminal 100 amino acids of Orc1 to be important for silencing, since truncations within this region impaired silencing. The deletion of 51 amino acids from the Orc1 N-terminus interrupted the interaction with Sir1 and also reduced silencing in sir1∆ strains. We thus propose that the silencing function of Orc1 is not restricted to Sir1 recruitment, but also comprises the interaction with another protein. The silencing function of this hypothesized interaction partner may depend on the N α-acetylation and integrity of the N-terminus of Orc1. In summary, we propose that N α-acetylation by NatA represents a protein modification that modulates chromatin structure in yeast.