Local opening of the DNA double helix in eukaryotic cells detected by osmium probe and adduct-specific immunofluorescence

Abstract

The structure of DNA in mouse fibroblast 3T3 cells has been investigated with the single-strand-selective probe, the complex of osmium tetroxide and 2,2'-bipyridine (Os,bipy). DNA-Os,bipy adducts in the cells were detected by immunofluorescence using a highly specific, affinity-purified polyclonal antibody. Treatment of living cells with the chemical probe led to a distinct but nonuniform nuclear staining. We attribute the positive nuclear staining to the existence of single-stranded and distorted DNA regions in the living cell. Confocal laser scanning microscopy revealed dark areas corresponding to nucleoli and regions of condensed chromatin. These conclusions were supported by the results of experiments in which the chemical probe was applied to fixed cells treated with 45% acetic acid or with acidic buffers (pH 1.8-2.5). An inverse staining pattern was obtained, characterized by intense immunofluorescence of the condensed chromatin regions. Thus, the structural transitions and/or chemical alterations (e.g. depurination) induced by acid treatment increase the accessibility of normally unreactive DNA bases. We conclude that open DNA structures recognized by the chemical probe in the cells prior to their fixation are contained mainly in decondensed and transcriptionally active chromatin, but are virtually absent from nucleoli and condensed chromatin.