A simple, sensitive assay to detect DNA-protein crosslinks in intact cells and in vivo.
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.
Zhitkovich
A
A
New York University Medical Center, Nelson Institute of Environmental Medicine, NY 10016.
Costa
M
M
D000818:Animals; D002857:Chromium / Q000494:pharmacology; D002945:Cisplatin / Q000494:pharmacology; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000032:analysis / Q000187:drug effects*; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D005557:Formaldehyde / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
D003432:Cross-Linking Reagents; D002857:Chromium; D005557:Formaldehyde; D012967:Sodium Dodecyl Sulfate; D011189:Potassium Chloride; D004247:DNA; D002945:Cisplatin