Dependence of the structure and mechanics of metaphase chromosomes on oxidized cysteines

Chromosome Res. 2016 Sep;24(3):339-53. doi: 10.1007/s10577-016-9528-6. Epub 2016 May 5.

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.

Keywords: Chromosome structure; Cysteine; Disulfide; Metaphase chromosome.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adenosine Triphosphatases / metabolism
  • Animals
  • Cell Cycle Proteins / metabolism
  • Cell Line
  • Chromosomal Proteins, Non-Histone / chemistry*
  • Chromosomal Proteins, Non-Histone / metabolism
  • Chromosomes, Human / chemistry*
  • Chromosomes, Human / ultrastructure*
  • Cohesins
  • Cysteine / chemistry*
  • DNA-Binding Proteins / metabolism
  • Dithiothreitol / chemistry
  • Electrophoresis, Gel, Two-Dimensional
  • Guanine Nucleotide Exchange Factors / metabolism
  • HEK293 Cells
  • Humans
  • Karyotype
  • Mass Spectrometry
  • Metaphase / genetics*
  • Micromanipulation
  • Multiprotein Complexes / metabolism
  • Notophthalmus viridescens / genetics
  • Nuclear Matrix-Associated Proteins / metabolism
  • Nuclear Proteins / metabolism
  • Octamer Transcription Factors / metabolism
  • Oxidation-Reduction
  • Phosphines / chemistry
  • RNA-Binding Proteins / metabolism

Substances

  • Cell Cycle Proteins
  • Chromosomal Proteins, Non-Histone
  • DNA-Binding Proteins
  • Guanine Nucleotide Exchange Factors
  • Multiprotein Complexes
  • NONO protein, human
  • Nuclear Matrix-Associated Proteins
  • Nuclear Proteins
  • Octamer Transcription Factors
  • Phosphines
  • RCC1 protein, human
  • RCC2 protein, human
  • RNA-Binding Proteins
  • condensin complexes
  • tris(2-carboxyethyl)phosphine
  • Adenosine Triphosphatases
  • Cysteine
  • Dithiothreitol