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.
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This work was supported by the NSF through grants MCB-1022117 and DMR-1206868 and by the NIH through grants R01-GM105847 and U54-CA193419 and by subcontract to U54-DK107980. Work at the Northwestern Proteomics Core was supported in part by the Northwestern Office of Research and the Feinberg School of Medicine.
Compliance with ethical standards
The experiments described in this article comply with the current laws of the country where they were performed (USA). This article does not contain any studies with human or animal subjects performed by any of the authors.
Conflicts of interest
The authors declare that they have no conflicts of interest.
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