| All eukaryotic cells proliferate by the faithful segregation of their genetic material to the daughter cells. This process is accomplished by attachment of a bipolar spindle structure to discrete, and dynamic macromolecular complexes that assemble on the centromeric region of replicated chromosomes known as kinetochores. Kinetochore function in budding yeast can be regulated by placing an inducible promoter adjacent to the centromeric DNA and integrated into the chromosome to create a conditional dicentric chromosome. Dicentric chromosomes undergo a breakage-fusion-bridge cycle as a consequence of having two centromeres on the same chromatid attach to opposite spindle poles in mitosis. Suppression of dicentric chromosome breakage reflects loss of kinetochore function at the kinetochore-microtubule or the kinetochore-DNA interface. I have utilized the conditionally functional dicentric chromosome in vivo, to demonstrate that kinetochore mutants exhibit quantitative differences in their degree of chromosome breakage. One class, represented by ctf17 /chl4/mcm17, completely suppresses dicentric chromosome breakage. Dicentric chromosomes are segregated without rearrangements in chl4Delta cells, indicating that only one of the two centromeres is functional. The study demonstrates that de novo centromere function is distinct from propagation of pre-existing centromeres. Following kinetochore assembly, accurate segregation of sister chromatids depends on achieving bi-orientation and maintaining kinetochore microtubule attachments during anaphase. A second class is represented by the conserved microtubule plus end binding protein Bim1p (yEB1). Human EB1 interacts with adenomatous polyposis coli (APC) and this interaction is often disrupted in colon cancers. I find that Bim1p (yEB1) is required for cellular viability of dicentric chromosome containing cells. Reduction in viability in bim1 mutant cells can be attributed to increased malsegregation of the dicentric chromosome and implicates Bim1p in being required for proper kinetochore-microtubule attachments in mitosis. These studies extend the use of the dicentric chromosome as an in vivo tool to study mutations in genes affecting discrete aspects of kinetochore function. |
