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.
Duke Scholars
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- Tumor Cells, Cultured
- Single-Strand Specific DNA and RNA Endonucleases
- Sensitivity and Specificity
- Nucleic Acid Renaturation
- Nucleic Acid Denaturation
- Humans
- Fluorescence
- Ethidium
- DNA, Single-Stranded
- Cross-Linking Reagents
Citation
Published In
DOI
ISSN
Publication Date
Volume
Issue
Start / End Page
Location
Related Subject Headings
- Tumor Cells, Cultured
- Single-Strand Specific DNA and RNA Endonucleases
- Sensitivity and Specificity
- Nucleic Acid Renaturation
- Nucleic Acid Denaturation
- Humans
- Fluorescence
- Ethidium
- DNA, Single-Stranded
- Cross-Linking Reagents