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Vol. 12, Issue 9, 2601-2613, September 2001
and
*Department of Molecular and Cell Biology, University of
California, Berkeley, California 94720-3202; and
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.
Memorial Sloan-Kettering Cancer Center, New York, New
York 10021
These authors contributed equally to this work.
§
Corresponding author. E-mail address:
gbarnes{at}socrates.berkeley.edu.
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