Effects of 5-bromo-2′-deoxyuridine and dimethyl sulfoxide on properties and structure of chromatin

Abstract We have investigated and compared the physico-chemical properties of chromatin that was isolated from Krebs II murine ascites tumor cells propagated either in the absence or presence of varying doses of 5-bromo-2′-deoxyuridine. These chromatins are shown to be altered as a result of BrdUrd ‡ substitution in the chromatin DNA when studied by thermal denaturation, circular dichroic spectropolarimetry, ethidium bromide binding, and by fractionation on ECTHAM-cellulose (Peterson & Kuff, 1969). The thermostability of chromatin is found to increase with BrdUrd substitution. On the other hand, thermomelting in the presence of 1% dimethyl sulfoxide indicates a decrease in thermostability of all base-pairs, as well as a destabilizing effect on protein-DNA interactions. The circular dichroism spectra of BrdUrd chromatin exhibit a 4 to 16% decrease in positive-band ellipticity at 277 nm. Concentrations of Me 2 SO up to 5% induce in these chromatins positive shifts in ellipticity towards B -DNA conformation of no more than 10% of base-pairs if two discrete conformational states are assumed. Scatchard plots of spectrophotometric titration of chromatin with ethidium bromide indicate a further reduction of primary binding sites in BrdUrd chromatin. BrdUrd effects on the number of binding sites or dye affinity in DNA, however, are not observed for intercalative binding, but are observed for the complex II binding mode. Furthermore, ECTHAM-cellulose chromatography reveals that the BrdUrd-containing chromatin fragments elute at a higher pH than unsubstituted fragments. The shift to a more acidic nature in BrdUrd-containing fragments may be accounted for by a shift in the non-histone/histone protein ratio, possibly arising from metabolic changes or modification of specific protein-DNA binding favoring non-histone proteins. These results are compatible with the hypothesis that BrdUrd promotes localized changes in chromatin condensation, probably by modification of chromosomal protein-DNA interactions, although the possibility that chromosomal proteins are modified, is not at present ruled out. In contrast, dimethyl sulfoxide antagonizes the effects produced by BrdUrd substitution by allowing chromatin to transform to a more relaxed (extended) state.