STRUCTURAL ANALYSIS OF A EUKARYOTIC CENTROMERE

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Centromeres of eukaryotic chromosomes are specific regions along the chromatin fiber that play a fundamental role in chromosome movement. Centromere DNA sequences isolated from the yeast, Saccharomyces cerevisiae, enable foreign DNA introduced into yeast to function as ordinary yeast chromosomes during cell division. We will examine how the centromere DNA sequence interacts with chromatin components in the cell nucleus, including histone and non-histone proteins. We have been able to selectively excise the centromere from yeast cells and affinity purify the protein-DNA complex. This methodology provides a unique opportunity to identify proteins bound to centromere DNA in vivo. we will examine how the kinetochore components are assembled onto the DNA, and when the complex attaches to the mitotic apparatus. The ability to conditionally regulate centromere function by transcription from an adjacent promoter allows the precise genetic control of chromosome movement during the cell cycle. We will study when in the cell cycle chromosomes are subject to mitotic forces and how the cell monitors the fidelity of chromosome segregation. The conditional centromere and selected point mutations in centromere DNA are indistinguishable in their loss of function, but differ in the mechanism of inactivation. We can discriminate loss of protein binding at the centromere from mutations in protein-protein interactions required for centromere function, and will identify mutants in protein binding to centromere DNA. The identification of trans-acting factors required for chromosome movement will enable us to determine how these factors interact with the centromere DNA to give rise to a functional unit responsible for chromosome locomotion. We will determine the role of a tightly centromere-linked gene, SP015 in spindle morphogenesis. SP015 is a member of a new class of GTP-binding proteins, that are microtubule-based mechanochemical enzymes. Knowledge of the factors that control the organization of the centromere and spindle may provide fundamental principles governing the molecular mechanism of cell division.