A SIMPLE, SENSITIVE ASSAY TO DETECT DNA-PROTEIN CROSS-LINKS IN INTACT-CELLS AND INVIVO

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, cisplatinum 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.