title: Reconstitution of mitotic chromatids with a minimum set of purified factors.
abstract: The assembly of mitotic chromosomes, each composed of a pair of rod-shaped chromatids, is an essential prerequisite for accurate transmission of the genome during cell division. It remains poorly understood, however, how this fundamental process might be achieved and regulated in the cell. Here we report an in vitro system in which mitotic chromatids can be reconstituted by mixing a simple substrate with only six purified factors: core histones, three histone chaperones (nucleoplasmin, Nap1 and FACT), topoisomerase II (topo II) and condensin I. We find that octameric nucleosomes containing the embryonic variant H2A.X-F are highly susceptible to FACT and function as the most productive substrate for subsequent actions of topo II and condensin I. Cdk1 phosphorylation of condensin I is the sole mitosis-specific modification required for chromatid reconstitution. This experimental system will enhance our understanding of the mechanisms of action of individual factors and their cooperation during this process.
authors.affiliation: Chromosome Dynamics Laboratory, RIKEN, Wako, Saitama 351-0198, Japan.
authors.forename: Keishi
authors.initials: K
authors.lastname: Shintomi
authors.affiliation: Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
authors.forename: Tatsuro S
authors.initials: TS
authors.lastname: Takahashi
authors.affiliation: Chromosome Dynamics Laboratory, RIKEN, Wako, Saitama 351-0198, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Hirano
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002842:Chromatids / Q000201:enzymology*; D042002:Chromatin Assembly and Disassembly; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D006367:HeLa Cells; D006609:High Mobility Group Proteins / Q000378:metabolism; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008297:Male; D008938:Mitosis; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000378:metabolism; D056489:Nucleoplasmins / Q000378:metabolism; D009707:Nucleosomes / Q000201:enzymology; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013094:Spermatozoa / Q000201:enzymology*; D035602:Transcriptional Elongation Factors / Q000378:metabolism; D014162:Transfection; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D006657:Histones; D018832:Molecular Chaperones; D046912:Multiprotein Complexes; D056489:Nucleoplasmins; D009707:Nucleosomes; C073537:SSRP1 protein, human; D029701:Saccharomyces cerevisiae Proteins; D035602:Transcriptional Elongation Factors; D029867:Xenopus Proteins; C108312:condensin complexes; D016203:CDC2 Protein Kinase; D000251:Adenosine Triphosphatases; D004250:DNA Topoisomerases, Type II