title: Preparation of Recombinant Centromeric Nucleosomes and Formation of Complexes with Nonhistone Centromere Proteins.
abstract: Centromeres are present on each chromosome to direct proper segregation during cell division. The understanding of how the histone H3 variant, CENP-A, epigenetically marks the location of the centromere on the chromosome has been advanced, in part, through the study of histone complexes, nucleosomes, and nucleosomal complexes with nonhistone centromere proteins. In this chapter, we describe the preparation of recombinant versions of these complexes. The methodology is firmly rooted in classic nucleosome reconstitution methods, but we highlight the aspects of the preparations that diverge from those used for the methods established with canonical histones. We also provide a method for producing PCR-amplified nucleosomal DNA sequences in milligram quantities that is particularly useful for studies where multiple sequences and/or chemical modifications are desired. Lastly, we describe our approach to assemble and analyze a complex between the recombinant human CENP-A nucleosome and one of its binding partners, CENP-C.
authors.affiliation: Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States.
authors.forename: N
authors.initials: N
authors.lastname: Sekulic
authors.affiliation: Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States. Electronic address: blackbe@mail.med.upenn.edu.
authors.forename: B E
authors.initials: BE
authors.lastname: Black
mesh_terms: D001324:Autoantigens / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002503:Centromere / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006657:Histones / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D009707:Nucleosomes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011485:Protein Binding; D017510:Protein Folding; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001324:Autoantigens; C000619700:CENPA protein, human; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone; D006657:Histones; D009707:Nucleosomes; D011994:Recombinant Proteins; C060905:centromere protein C; D004247:DNA
keywords: CENP-A; Centromere; Chromosome; Histone; Kinetochore; Nucleosome; Reconstitution