title: A cell-free system for functional centromere and kinetochore assembly.
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.
authors.affiliation: Department of Biochemistry, Stanford Medical School, Stanford, California, USA.
authors.forename: Annika
authors.initials: A
authors.lastname: Guse
authors.forename: Colin J
authors.initials: CJ
authors.lastname: Fuller
authors.forename: Aaron F
authors.initials: AF
authors.lastname: Straight
mesh_terms: D000818:Animals; D001324:Autoantigens / Q000737:chemistry / Q000378:metabolism; D002474:Cell-Free System; D002503:Centromere / Q000378:metabolism* / Q000648:ultrastructure; D000076247:Centromere Protein A; D002843:Chromatin / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000378:metabolism; D005455:Fluorescent Antibody Technique / Q000379:methods; D018386:Kinetochores / Q000378:metabolism* / Q000648:ultrastructure; D014981:Xenopus
chemical_list: D001324:Autoantigens; D000076247:Centromere Protein A; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone