Yeast Dam1p has a role at the kinetochore in assembly of the mitotic spindle

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Abstract
During mitosis, replicated chromosomes are separated to daughter cells by the microtubule-based mitotic spindle. Chromosomes attach to the mitotic spindle through specialized DNA/protein structures called kinetochores, but the mechanism of attachment is not well understood. We show here that the yeast microtubule-binding protein, Dam1p, associates physically and functionally with kinetochores, suggesting a role in kinetochore attachment to the spindle. An epitope-tagged version of Dam1p colocalizes with the integral kinetochore component Ndc10p/Cbf2p in immunofluorescence analysis of chromosome spreads. In addition, Dam1p is associated preferentially with centromeric DNA as shown by chromatin immunoprecipitation experiments, and this association depends on Ndc10p/Cbf2p. We also demonstrate genetic interactions between DAM1 and CTF19 or SLK19 genes encoding kinetochore proteins. Although the defect caused by the dam1-1 mutation leads to activation of the spindle checkpoint surveillance system and consequent persistence of sister chromatid cohesion, the metaphase arrest spindle abnormally elongates, resulting in virtually complete chromosome missegregation. Execution point experiments indicate that Dam1p has a role in formation of a metaphase spindle and in anaphase spindle elongation. Finally, we have observed that the protein encoded by the dam1-1 allele becomes delocalized at the nonpermissive temperature, correlating with the subsequent onset of the mutant phenotype. Our studies are consistent with a role for Dam1p in attachment of sister chromatids through the kinetochore to the mitotic spindle before chromosome segregation.