Dad1p, third component of the Duo1p/Dam1p complex involved in kinetochore function and mitotic spindle integrity.
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.
Enquist-Newman
M
M
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202, USA.
Cheeseman
I M
IM
Van Goor
D
D
Drubin
D G
DG
Meluh
P B
PB
Barnes
G
G
D000483:Alleles; D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism; D020090:Chromosome Segregation / Q000187:drug effects; D003598:Cytoskeletal Proteins; D004843:Epistasis, Genetic; D015966:Gene Expression Regulation, Fungal; D018386:Kinetochores / Q000187:drug effects / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D015739:Nocodazole / Q000494:pharmacology; D011233:Precipitin Tests; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D008941:Spindle Apparatus / Q000187:drug effects / Q000378:metabolism*; D013696:Temperature; D020798:Two-Hybrid System Techniques
D018797:Cell Cycle Proteins; D002843:Chromatin; D003598:Cytoskeletal Proteins; C116003:DAM1 protein, S cerevisiae; C116004:DUO1 protein, S cerevisiae; C439311:Dad1 protein, S cerevisiae; D008869:Microtubule-Associated Proteins; D029701:Saccharomyces cerevisiae Proteins; D015739:Nocodazole