Characterization of the role of serine phosphorylation on CenH3 function in Saccharomyces cerevisae

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Centromeres are defined regions of the chromosomes that direct the assembly of the kinetochores, which ensure proper segregation of sister chromatids during cell division. The centromeric DNA of Saccharomyces cerevisiae is packaged in a single nucleosome containing the centromeric histone H3 variant Cse4 in place of the usual histone H3. In work preceding this proposal, we have identified a novel posttranslational modification (PTM) of Cse4, the phosphorylation of serine 33. Here, we will determine the functional significance of this modification. To this end, genetic analysis of mutation of this site to alanine will be performed, and its effect on cell cycle progression, kinetochore assembly and chromosome segregation will be determined. Furthermore, a serine 33 phosphorylation-specific antibody will be generated, and the presence of modified free and chromatin-bound Cse4 will be determined. Finally, this antibody will be used to search for the enzyme responsible for this modification. These studies of a novel modification on the centromeric histone variant will allow important new insights into the regulation of centromere function by PTMs and thus will open up a new avenue of investigation that so far has not been explored.