A simple, sensitive assay to detect DNA–protein cromlinks in intact cells and in vivo

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Abstract
Addition of potassium chloride to sodium dodecyl sulfate (SDS) resulted in the formation of an insoluble precipitate that was easily recovered by low-speed centrifugation. Since SDS tightly binds to proteins but not DNA, all proteins and detergent-resistant DNA--protein complexes were also effectively co-precipitated in the presence of potassium--SDS leaving free DNA in the supernatant. The amount of SDS-precipitable DNA represented a measure of DNA--protein crosslinks. We have adapted this method for determining DNA--protein crosslinks formed in cells following their exposure in culture or in vivo to crosslinking agents such as chromate, cis-Pt(II) diammine dichloride and formaldehyde. The critical parameters for application of the K--SDS assay to cells were rigorously reproducible shearing of chromosomal DNA and effective washing steps. We have found that freeze-thawing SDS lysed cells followed by vortexing and repeated resuspensions of the precipitate by pipeting resulted in a low background and high reproducibility of the assay. The method detected in a dose-dependent manner DNA--protein crosslinks induced in CHO cells by chromate, cis-platinum and formaldehyde, with sensitivity similar to the alkaline elution procedure. The K--SDS assay was also successfully utilized to determine DNA--protein crosslinks in rat and mouse white blood cells exposed in vivo to chromate. Its sensitivity and simplicity in sample handling and DNA--protein complex isolation potential allows wide application of the assay to measure formation of DNA--protein crosslinks. The ease of recovery of DNA--protein complexes allows for a more thorough investigation of this lesion.

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