Measurement of DNA-protein crosslinks in mammalian cells without X-irradiation.
To study the mechanisms of formation and repair of DNA-protein crosslinks in mammalian cells, the best general method to assay these lesions is the Kohn membrane alkaline elution procedure. Use of this sensitive technique requires the introduction of random strand breaks in the DNA by X-irradiation to reduce the very high molecular weight so that it elutes off the filter at an appropriate rate. This report describes an alternative method for fragmenting the DNA in the absence of X-irradiation equipment. Convenient reproducible elution rates of DNA from various mouse and human cells in culture without X-irradiation result from elution through polyvinyl chloride filters with 75 mM sodium hydroxide (0.33 ml/min) instead of the standard 20 mM EDTA-tetrapropylammonium hydroxide, pH 12.2 (0.03 to 0.04 ml/min). Dose-dependent retardation of the DNA elution was observed over the range 0 to 30 microM trans-platinum(II)diamminedichloride, and proteinase K treatment during cell lysis restored the elution rate to that of the untreated control cell DNA. In the absence of X-irradiation, this elution method measures DNA-protein crosslinks with higher sensitivity and equivalent reproducibility as the air-burst procedure.
D000818:Animals; D002478:Cells, Cultured; D002945:Cisplatin; D004247:DNA / Q000032:analysis* / Q000528:radiation effects; D004273:DNA, Neoplasm / Q000032:analysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007939:Leukemia L1210 / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D011485:Protein Binding; D012972:Sodium Hydroxide
D004273:DNA, Neoplasm; C034697:transplatin; D012972:Sodium Hydroxide; D004247:DNA; D002945:Cisplatin