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

Molecular toxicology Pub Date : 1989-01-01
C A Miller, M Costa
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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.

用甲醛、铬酸盐和顺式二胺二氯铂处理细胞后与DNA交联的蛋白质分析(II)。
通过二维(2D)凝胶电泳、免疫印迹和离心分析来比较暴露于顺式二胺二氯铂(cis-Pt)、铬酸盐和甲醛后培养的中国仓鼠卵巢细胞与DNA交联的蛋白质。铬酸盐和顺式铂将7种相同的非组蛋白(如肌动蛋白)与DNA交联。相反,甲醛选择性地形成组蛋白- dna交联。免疫印迹实验表明,尽管这三种化学物质与DNA和蛋白质的化学反应性不同,但它们都能将一个97 kda的核蛋白与DNA交联。铬酸盐和顺式pt诱导的交联被硫脲、2-巯基乙醇和EDTA破坏,表明金属可能从交联中被化学置换。甲醛诱导的复合物需要用DNase 1降解才能在2D凝胶上分解组蛋白,并且不像金属诱导的交联那样具有化学不稳定性。这里使用的试剂和方法可以应用于研究结合或驻留在DNA附近的其他核蛋白。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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