RESUMO
We showed recently that a complex between Duo1p and Dam1p is required for both spindle integrity and kinetochore function in the budding yeast Saccharomyces cerevisiae. To extend our understanding of the functions and interactions of the Duo1p/Dam1p complex, we analyzed the novel gene product Dad1p (for Duo1 and Dam1 interacting). Dad1p physically associates with Duo1p by two-hybrid analysis, coimmunoprecipitates with Duo1p and Dam1p out of yeast protein extracts, and shows interdependent localization with Duo1p and Dam1p to the mitotic spindle. These results indicate that Dad1p functions as a component of the Duo1p/Dam1p complex. Like Duo1p and Dam1p, Dad1p also localizes to kinetochore regions in chromosomes spreads. Here, we also demonstrate by chromatin immunoprecipitation that Duo1p, Dam1p, and Dad1p associate specifically with centromeric DNA in a manner that is dependent upon Ndc10 and partially dependent upon the presence of microtubules. To explore the functions of Dad1p in vivo, we generated a temperature-sensitive allele, dad1-1. This allele shows spindle defects and a mitotic arrest phenotype that is dependent upon the spindle assembly checkpoint. In addition, dad1-1 mutants undergo chromosome mis-segregation at the restrictive temperature, resulting in a dramatic decrease in viability.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Fuso Acromático/metabolismo , Alelos , Sequência de Aminoácidos , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Segregação de Cromossomos/efeitos dos fármacos , Proteínas do Citoesqueleto , Epistasia Genética , Regulação Fúngica da Expressão Gênica , Cinetocoros/efeitos dos fármacos , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Mutação/genética , Nocodazol/farmacologia , Testes de Precipitina , Ligação Proteica , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Fuso Acromático/efeitos dos fármacos , Temperatura , Técnicas do Sistema de Duplo-HíbridoRESUMO
Duo1p and Dam1p were previously identified as spindle proteins in the budding yeast, Saccharomyces cerevisiae. Here, analyses of a diverse collection of duo1 and dam1 alleles were used to develop a deeper understanding of the functions and interactions of Duo1p and Dam1p. Based on the similarity of mutant phenotypes, genetic interactions between duo1 and dam1 alleles, interdependent localization to the mitotic spindle, and Duo1p/Dam1p coimmunoprecipitation from yeast protein extracts, these analyses indicated that Duo1p and Dam1p perform a shared function in vivo as components of a protein complex. Duo1p and Dam1p are not required to assemble bipolar spindles, but they are required to maintain metaphase and anaphase spindle integrity. Immunofluorescence and electron microscopy of duo1 and dam1 mutant spindles revealed a diverse variety of spindle defects. Our results also indicate a second, previously unidentified, role for the Duo1p/Dam1p complex. duo1 and dam1 mutants show high rates of chromosome missegregation, premature anaphase events while arrested in metaphase, and genetic interactions with a subset of kinetochore components consistent with a role in kinetochore function. In addition, Duo1p and Dam1p localize to kinetochores in chromosome spreads, suggesting that this complex may serve as a link between the kinetochore and the mitotic spindle.
