The long range objective of this research is to understand the molecular mechanisms involved in the proper segregation of chromosomes to parent and daughter cells during mitosis and meiosis in eukaryotes. A critical chromosomal constituent of this key aspect of the cell division process, and the topic of this proposal, is the centromere, defined as a point of constriction where chromosome arms meet and where spindle attachment is mediated. We are analyzing the structural organization and defining the functional parameters of the centromere regions of each of the three chromosomes of Schizosaccharomyces pombe. Our goal is to identify the components that are needed both in cis and in trans for proper centromere function in fission yeast. The centromere regions in S. pombe are considerably more complex than those of the budding yeast Saccharomyces cerevisiae, and resemble those of higher eukaryotes in containing several classes of untranscribed repeated DNA sequences that encompass many kilobases of DNA on each chromosome. Thus, the S. pombe system, which is very amenable to biochemical and genetic analysis, is a particularly relevant model for higher eukaryotic centromere structure and function.
Our specific aims are to complete the detailed structural analysis of each of the three S. pombe centromere DNAs, with regard to the overall sizes of the regions, the organization of centromere-specific repeats, and the nucleotide sequence of the central core and core-associated repeat domains; and to determine the structural conformations and minimal DNA sequences required in cis to specify various centromere functions, including mitotic stability, 2+:2-meiotic segregation, and maintenance of sister chromatid attachment in meiosis I. Additionally, a major goal is directed towards the identification of components needed in trans for proper centromere function in S. pombe. These include the kinetochore proteins that play a direct role in the mechanics of centromere action, as well as those proteins involved in the regulation of centromere function. In order to identify protein binding sites in centromere DNA, our examination of centromeric chromatin will be extended to the mapping of DNaseI hypersensitive sites, which can be indicative of protein binding sites, within the central core regions and K"""""""" repeats that have been shown to be necessary for centromere function. The direct isolation of centromere DNA binding proteins will be attempted by standard biochemical fractionation techniques with a DNA fragment mobility shift assay or an antibody assay with antisera directed against S. cerevisiae centromeric proteins such as CBF3. Genes specifying centromeric proteins will be isolated, and null and conditional alleles constructed to study functional roles. Our minichromosome assay for centromere function provides a system for combining molecular and genetic approaches to identify and isolate other genes whose products are components of the centromere or are involved in regulation of its function.
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