Biochemical and Structural Analysis of Kinetochore Histone-Fold Complexes
The kinetochore connects chromosomes to microtubules during mitosis and therefore plays an essential role in faithful chromosome segregation. It is built at the centromeric region of the chromosome and is comprised of many protein complexes. CENP-S, -T, -W, and -X are kinetochore components with histone-folds. These proteins play important roles in establishment of kinetochore chromatin. Similar to canonical histones, these kinetochore histone-fold proteins form heteromeric complexes (CENP-S/CENP-X complex and CENP-T/CENP-W complex) and bind DNA in sequence independent manner. In addition, they form a CENP-T-W-S-X heterotetrameric complex and bind DNA in a manner that is different from both CENP-S-X and CENP-T-W. To understand how kinetochores form and function it is necessary to characterize the components in detail. Here, we describe our approaches in purification and characterization of the kinetochore histone-fold complexes.
Key wordsChromosome segregation Kinetochore Centromere Histone-fold Protein complex Protein expression Protein purification Protein–protein interaction Protein–DNA interaction Gel shift assay
We thank members of the Fukagawa lab in particular Kozo Takeuchi for his help with sample preparation and characterization. We also acknowledge Kiyomi Kita and Mayumi Takahashi for their contributions.
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