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Related Experiment Videos

Implication of a novel multiprotein Dam1p complex in outer kinetochore function.

I M Cheeseman1, C Brew, M Wolyniak

  • 1Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.

The Journal of Cell Biology
|January 5, 2002
PubMed
Summary
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The Dam1p complex, including newly identified proteins, is crucial for spindle integrity and kinetochore function in budding yeast mitosis. This complex directly binds microtubules, facilitating chromosome-spindle attachment.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Genetics

Background:

  • The Dam1p complex, comprising Dam1p, Duo1p, and Dad1p, is known to be essential for spindle integrity and kinetochore function in budding yeast.
  • Understanding the full composition and regulation of this complex is vital for elucidating mitotic mechanisms.

Purpose of the Study:

  • To purify and characterize the complete Dam1p complex.
  • To investigate the regulatory mechanisms and functional roles of the Dam1p complex and its subunits in mitosis.

Main Methods:

  • Purification of the Dam1p complex from yeast extracts.
  • Biochemical assays to determine microtubule binding affinity.
  • Localization studies using fluorescently tagged proteins (GFP).
  • Generation and analysis of temperature-sensitive mutants for novel subunits.

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Main Results:

  • A ~245 kD Dam1p complex was purified, containing previously known subunits (Dam1p, Duo1p, Dad1p) and newly identified proteins (Spc19p, Spc34p, Dad2p, Ask1p).
  • The complex is regulated by phosphorylation, with cell cycle-dependent changes observed.
  • The purified complex exhibits direct in vitro binding to microtubules with a 0.5 microM affinity.
  • Localization studies confirmed the presence of new subunits at the mitotic spindle and kinetochores.
  • Functional importance was demonstrated through temperature-sensitive mutants of DAD2 and ASK1.

Conclusions:

  • The Dam1p complex, including the four newly identified subunits, plays a critical role in outer kinetochore function.
  • The complex is well-positioned to mediate the crucial interaction between chromosomes and spindle microtubules during mitosis.
  • Phosphorylation likely serves as a key regulatory mechanism for the Dam1p complex's function throughout the cell cycle.