Regulators of Centromeric Nucleosomes in Saccharomyces cerevisiae

Shivaraju, Manjunatha (2011). Regulators of Centromeric Nucleosomes in Saccharomyces cerevisiae. PhD thesis The Open University.



The centromere is a specialized chromosomal structure that regulates faithful chromosome segregation during cell division, as it dictates the site of assembly of the kineto-chore. In all organisms, the centromeric nucleosomes are specified by a H3 variant, known as Cse4 in budding yeast. How these centromeric nucleosomes are assembled and perpetuated is only beginning to be understood. Scm3 is an evolutionarily conserved essential inner kinetochore protein that has been shown to be important for centromere specification. Plasmid supercoiling assays performed in vitro with recombinant proteins demonstrate that Scm3 can act as a Cse4-specific nucleosome assembly factor. Assembly activity depends on an evolutionarily conserved domain of Scm3 and the centromere targeting domain (CATD) of Cse4, but is sequence independent. Interestingly, micrococcal nuclease digestion of Scm3 assembled Cse4 nucleosomes reveals that less DNA is protected compared to Nap 1 assembled Cse4 nucleosomes, suggesting structural differences. Fluorescence correlation spectroscopy in combination with brightness measurements and confocal imaging experiments in live cells revealed that centromeres at G1 phase have one copy of Cse4 per centromeric nucleosome whereas two copies are detected at anaphase. The apparent structural change occurs at the “metaphase to anaphase” transition. We propose a model in which the structure and composition of centromeric nucleosomes is cell cycle regulated. Our model reconciles experimental evidence for the existence of both the hemisome and the octasome.

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