S1-nuclease enhancement of the ethidium bromide binding assay of drug-induced DNA interstrand crosslinking in human brain tumor cells.
A modification, using S1-nuclease, of a simple and sensitive fluorometric assay with ethidium bromide was developed for the measurement of cellular DNA interstrand crosslinking induced by bifunctional alkylators. Cells are lysed and treated with proteinase K and sodium dodecyl sulfate followed by extensive dialysis to yield intact high-molecular-weight DNA, free of contaminating proteins, on which the crosslink assay is then performed. The assay depends on the differential binding of ethidium bromide to single- and double-stranded DNA. Because of the higher ethidium bromide binding capacity of double-stranded DNA, the fluorescence retained after a heating/cooling cycle is directly proportional to the drug-induced cellular DNA interstrand crosslinking. We demonstrate that the sensitivity of this assay can be increased up to fourfold by including an S1-nuclease digestion step. This modified technique is simple and suited to the quantitation of low levels of DNA-interstrand crosslinking in cells.
Sriram
R
R
Department of Neurological Surgery, University of Washington Medical Center, Seattle 98195.
Ali-Osman
F
F
D000477:Alkylating Agents / Q000493:pharmacokinetics*; D001932:Brain Neoplasms / Q000235:genetics*; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000493:pharmacokinetics*; D004277:DNA, Single-Stranded / Q000187:drug effects / Q000378:metabolism*; D004996:Ethidium / Q000493:pharmacokinetics*; D005453:Fluorescence; D006801:Humans; D009691:Nucleic Acid Denaturation; D009695:Nucleic Acid Renaturation; D012680:Sensitivity and Specificity; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014407:Tumor Cells, Cultured
D000477:Alkylating Agents; D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D015719:Single-Strand Specific DNA and RNA Endonucleases; D004996:Ethidium