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.