Analysis of proteins cross-linked to DNA after treatment of cells with formaldehyde, chromate, and cis-diamminedichloroplatinum(II).
The proteins cross-linked to the DNA of cultured Chinese hamster ovary cells after exposure to cis-diamminedichloroplatinum(II) (cis-Pt), chromate, and formaldehyde were compared by two-dimensional (2D) gel electrophoresis, immunoblotting, and centrifugal assays that measured cross-link stability. Chromate and cis-Pt cross-linked seven of the same nonhistone proteins, such as actin, to DNA. In contrast, formaldehyde selectively formed histone-DNA cross-links. Immunoblotting experiments showed that all three chemicals cross-linked a 97-kDa nuclear protein to the DNA despite their different chemical reactivity with DNA and proteins. The chromate- and cis-Pt-induced cross-links were disrupted by thiourea, 2-mercaptoethanol, and EDTA, indicating that the metal could be chemically displaced from the cross-links. The formaldehyde-induced complexes required degradation with DNase 1 for the resolution of histones on 2D gels and were not chemically labile like the metal-induced cross-links. The agents and methodology used here could be applied to the study of additional nuclear proteins that bind or reside near the DNA.
Miller
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CA
Institute of Environmental Medicine, New York University Medical Center, Tuxedo 10987.
Costa
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D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002840:Chromates / Q000494:pharmacology*; D002945:Cisplatin / Q000494:pharmacology*; D003432:Cross-Linking Reagents; D004247:DNA / Q000187:drug effects*; D015180:Electrophoresis, Gel, Two-Dimensional; D005557:Formaldehyde / Q000494:pharmacology*; D015151:Immunoblotting; D009687:Nuclear Proteins / Q000187:drug effects; D017680:Potassium Compounds; D011506:Proteins / Q000187:drug effects*; D014461:Ultracentrifugation
D002840:Chromates; D003432:Cross-Linking Reagents; D009687:Nuclear Proteins; D017680:Potassium Compounds; D011506:Proteins; D005557:Formaldehyde; C027373:potassium chromate(VI); D004247:DNA; D002945:Cisplatin