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Probing the Saccharomyces cerevisiae centromeric DNA (CEN DNA)-binding factor 3 (CBF3) kinetochore complex by using

L I Pietrasanta1, D Thrower, W Hsieh

  • 1Department of Physics, Cellular, and Developmental Biology, University of California, Santa Barbara, CA 93106, USA.

Proceedings of the National Academy of Sciences of the United States of America
|March 31, 1999
PubMed
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Yeast centromere DNA binding factor 3 (CBF3) complex binds centromeric DNA, shortening and bending it. This structure may hold chromatid pairs together during mitosis.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biophysics

Background:

  • Yeast centromeric DNA (CEN DNA) binding factor 3 (CBF3) is a crucial protein complex for accurate chromosome segregation.
  • CBF3 is composed of four proteins and is a core component of the yeast kinetochore, binding to the CDEIII element of CEN DNA.

Purpose of the Study:

  • To investigate the structural organization of the CBF3-CEN DNA complex using atomic force microscopy.
  • To elucidate the DNA bending and binding characteristics of the CBF3 complex.

Main Methods:

  • Purified CBF3 proteins were combined with a CEN DNA fragment from yeast chromosome III.
  • Atomic force microscopy (AFM) was employed to visualize and analyze the structure of the CBF3-CEN DNA complexes.

Main Results:

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  • AFM images revealed DNA molecules with attached globular CBF3 bodies.
  • The DNA contour length was reduced by ~9% upon complex formation, indicating DNA winding.
  • CEN DNA was bent ~55 degrees at the CBF3 binding site.
  • CBF3 complexes were observed to link two DNA molecules, forming multi-armed structures.

Conclusions:

  • The CBF3-CEN DNA complex exhibits DNA shortening and significant bending, consistent with its role in chromosome segregation.
  • The ability of CBF3 to bind two DNA molecules suggests a potential role in holding sister chromatids together during mitosis.