Assembly of Centromeric Nucleosomes in Budding Yeast

Abstract

The nucleosomes at centromeres contain a specialized histone H3 variant (CENP-A in metazoans/Cse4 in budding yeast) that is essential for recruiting kinetochore proteins and ensuring proper segregation. The deposition of CENP-A/Cse4 at centromeres requires dedicated assembly factors and mechanisms. In contrast to the much longer, "regional" centromeres of most other eukaryotes, those of "point centromeres" in budding yeast have defined consensus sequences and comprise three “centromere-determining elements” (CDEs): CDEI, CDEII, and CDEIII. The CBF3 complex recognizes CDEIII, recruits Cse4, and chaperones the assembly of a centromeric nucleosome. Loss of CBF3 leads to mitotic arrest. In addition to the role of CBF3 in recruiting Cse4 to centromeres, there are mechanisms that prevent Cse4 misincorporation into chromosome arms, safeguarding against formation of dicentric chromosomes and genomic instability. One mechanism is ubiquitin-mediated degradation of mislocalized Cse4. Indeed, Psh1 is an E3 ubiquitin ligase found in budding yeast that binds Cse4 and promotes polyubiquitination. Loss of Psh1 results in Cse4 misincorporation into non-centromeric sites, including sites of DNA repair and promoters. In this thesis, we describe the structure of the CBF3 complex and propose a mechanism for centromere recognition and centromeric nucleosome assembly in the budding yeast, Kluyveromyces lactis. We also demonstrate that Saccharomyces cerevisiae Psh1 recognizes Cse4 that is misincorporated into nucleosomes. These studies provide molecular details of the mechanisms that restrict Cse4 localization and centromeric nucleosome assembly, in order to maintain proper kinetochore recruitment and faithful sister chromatid segregation.