Analysis of proteins cross-linked to DNA after treatment of cells with formaldehyde, chromate, and cis-diamminedichloroplatinum(II)

Abstract

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.