The role of the centromeric histone variant in kinetochore specification and assembly.
Kimberly A. Collins, Sue Biggins
Basic Sciences, FHCRC, 1100 Fairview Ave. N, Seattle, WA, 98109, USA
The accurate segregation of sister chromatids to daughter cells is critical for maintaining genomic stability. Chromosome segregation is directed by the kinetochore, the protein complex that assembles on centromeric DNA. One hallmark of all kinetochores is a conserved histone H3 variant, called Cse4p in budding yeast, that is incorporated into a specialized centromeric nucleosome. It is unknown whether Cse4 functions in nucleating kinetochore assembly, in biorientation, or microtubule attachment. To investigate Cse4 functions, we isolated dominant lethal CSE4 mutants by mutagenizing a CSE4 gene under the control of the inducible galactose promoter. Phenotype analysis classified the mutants into three classes. We further investigated the class of mutants in which both mutant and endogenous Cse4 incorporates into the chromatin and found that these mutants do not nucleate extra kinetochores, suggesting that Cse4 is not sufficient to nucleate kinetochore assembly. This led us to investigate the role of Cse4 in kinetochore assembly. Surprisingly, we found that in the absence of Cse4, several kinetochore subcomplexes can fully localize to the conditional centromere, suggesting that the essential function of Cse4 is not to nucleate kinetochore assembly. We are currently investigating the phenotype of a conditional degron allele of Cse4 to determine whether Cse4 functions in microtubule attachment or biorientation at the kinetochore.
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