title: Dependence of the structure and mechanics of metaphase chromosomes on oxidized cysteines.
abstract: We have found that reagents that reduce oxidized cysteines lead to destabilization of metaphase chromosome folding, suggesting that chemically linked cysteine residues may play a structural role in mitotic chromosome organization, in accord with classical studies by Dounce et al. (J Theor Biol 42:275-285, 1973) and Sumner (J Cell Sci 70:177-188, 1984a). Human chromosomes isolated into buffer unfold when exposed to dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). In micromanipulation experiments which allow us to examine the mechanics of individual metaphase chromosomes, we have found that the gel-like elastic stiffness of native metaphase chromosomes is dramatically suppressed by DTT and TCEP, even before the chromosomes become appreciably unfolded. We also report protein labeling experiments on human metaphase chromosomes which allow us to tag oxidized and reduction-sensitive cysteine residues. PAGE analysis using fluorescent labels shows a small number of labeled bands. Mass spectrometry analysis of similarly labeled proteins provides a list of candidates for proteins with oxidized cysteines involved in chromosome organization, notably including components of condensin I, cohesin, the nucleosome-interacting proteins RCC1 and RCC2, as well as the RNA/DNA-binding protein NONO/p54NRB.
authors.affiliation: Department of Molecular Biosciences, Northwestern University, Evanston, IL, 60208, USA.
authors.forename: Adrienne
authors.initials: A
authors.lastname: Eastland
authors.affiliation: Department of Molecular Biosciences, Northwestern University, Evanston, IL, 60208, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Hornick
authors.affiliation: Department of Molecular Biosciences, Northwestern University, Evanston, IL, 60208, USA.|Department of Physics and Astronomy, Northwestern University, Evanston, IL, 60208, USA.
authors.forename: Ryo
authors.initials: R
authors.lastname: Kawamura
authors.affiliation: Proteomics Core, Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, 60208, USA.
authors.forename: Dhaval
authors.initials: D
authors.lastname: Nanavati
authors.affiliation: Department of Molecular Biosciences, Northwestern University, Evanston, IL, 60208, USA. john-marko@northwestern.edu.|Department of Physics and Astronomy, Northwestern University, Evanston, IL, 60208, USA. john-marko@northwestern.edu.
authors.forename: John F
authors.initials: JF
authors.lastname: Marko
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry* / Q000378:metabolism; D002877:Chromosomes, Human / Q000737:chemistry* / Q000648:ultrastructure*; D003545:Cysteine / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004229:Dithiothreitol / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D059785:Karyotype; D013058:Mass Spectrometry; D008677:Metaphase / Q000235:genetics*; D008846:Micromanipulation; D046912:Multiprotein Complexes / Q000378:metabolism; D009673:Notophthalmus viridescens / Q000235:genetics; D034521:Nuclear Matrix-Associated Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D050810:Octamer Transcription Factors / Q000378:metabolism; D010084:Oxidation-Reduction; D010720:Phosphines / Q000737:chemistry; D016601:RNA-Binding Proteins / Q000378:metabolism; D000097722:Cohesins
chemical_list: D000251:Adenosine Triphosphatases; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D003545:Cysteine; D004268:DNA-Binding Proteins; D004229:Dithiothreitol; D020662:Guanine Nucleotide Exchange Factors; D046912:Multiprotein Complexes; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins; D050810:Octamer Transcription Factors; D010720:Phosphines; D016601:RNA-Binding Proteins; D000097722:Cohesins; C083214:NONO protein, human; C062521:RCC1 protein, human; C477650:RCC2 protein, human; C108312:condensin complexes; C080938:tris(2-carboxyethyl)phosphine
keywords: Chromosome structure; Cysteine; Disulfide; Metaphase chromosome