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Nat Protoc. 2012 Oct;7(10):1847-69. doi: 10.1038/nprot.2012.112. Epub 2012 Sep 20.

A cell-free system for functional centromere and kinetochore assembly.

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1
Department of Biochemistry, Stanford Medical School, Stanford, California, USA.

Abstract

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.

PMID:
23018190
PMCID:
PMC4011387
DOI:
10.1038/nprot.2012.112
[Indexed for MEDLINE]
Free PMC Article
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