{"title":"Analysis of proteins cross-linked to DNA after treatment of cells with formaldehyde, chromate, and cis-diamminedichloroplatinum(II).","authors":"C A Miller, M Costa","doi":"","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":77750,"journal":{"name":"Molecular toxicology","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"1989-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Molecular toxicology","FirstCategoryId":"1085","ListUrlMain":"","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
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.