A cell-free system for functional centromere and kinetochore assembly
This protocol describes a cell-free system for studying vertebrate centromere and kinetochore formation. We reconstitute tandem arrays of centromere protein A (CENP-A) nucleosomes as a substrate for centromere and kinetochore assembly. These chromatin substrates are immobilized on magnetic beads and...
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| Main Authors: | , , |
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| Format: | Article (Journal) |
| Language: | English |
| Published: |
20 September 2012
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| In: |
Nature protocols
Year: 2012, Volume: 7, Issue: 10, Pages: 1847-1869 |
| ISSN: | 1750-2799 |
| DOI: | 10.1038/nprot.2012.112 |
| Online Access: | Verlag, Volltext: http://dx.doi.org/10.1038/nprot.2012.112 Verlag, Volltext: https://www.nature.com/nprot/journal/v7/n10/full/nprot.2012.112.html 
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| Author Notes: | Annika Guse, Colin J. Fuller, Aaron F. Straight |
| Summary: | This protocol describes a cell-free system for studying vertebrate centromere and kinetochore formation. We reconstitute tandem arrays of centromere protein A (CENP-A) nucleosomes as a substrate for centromere and kinetochore assembly. These chromatin substrates are immobilized on magnetic beads and then incubated in Xenopus egg extracts that provide a source for centromere and kinetochore proteins and that can be cycled between mitotic and interphase cell cycle states. This cell-free system lends itself to use in protein immunodepletion, complementation and drug inhibition as a tool to perturb centromere and kinetochore assembly, cytoskeletal dynamics, DNA modification and protein post-translational modification. This system provides a distinct advantage over cell-based investigations in which perturbing centromere and kinetochore function often results in lethality. After incubation in egg extract, reconstituted CENP-A chromatin specifically assembles centromere and kinetochore proteins, which locally stabilize microtubules and, on microtubule depolymerization with nocodazole, activate the mitotic checkpoint. A typical experiment takes 3 d. |
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| Item Description: | Gesehen am 04.05.2017 |
| Physical Description: | Online Resource |
| ISSN: | 1750-2799 |
| DOI: | 10.1038/nprot.2012.112 |