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

During mitosis, replicated chromosomes are separated to daughter cells by t he microtubule-based mitotic spindle. Chromosomes attach to the mitotic spi ndle through specialized DNA/protein structures called kinetochores, but th e mechanism of attachment is not well understood. We show here that the yea st microtubule-binding protein, Dam1p, associates physically and functional ly with kinetochores, suggesting a role in kinetochore attachment to the sp indle. An epitope-tagged version of Dam1p colocalizes with the integral kin etochore component Ndc10p/Cbf2p in immunofluorescence analysis of chromosom e spreads. in addition, Dam1p is associated preferentially with centromeric DNA as shown by chromatin immunoprecipitation experiments, and this associ ation depends on Ndc10p/Cbf2p. We also demonstrate genetic interactions bet ween 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 chroma tid cohesion, the metaphase arrest spindle abnormally elongates, resulting in virtually complete chromosome missegregation. Execution point experiment s indicate that Dam1p has a role in formation of a metaphase spindle and in anaphase spindle elongation. Finally, we have observed that the protein en coded by the dam1-1 allele becomes delocalized at the nonpermissive tempera ture, correlating with the subsequent onset of the mutant phenotype. Our st udies are consistent with a role for Dam1p in attachment of sister chromati ds through the kinetochore to the mitotic spindle before chromosome segrega tion.