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Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae
Published on: June 14, 2013
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.
Summary
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:
- 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.
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