DNA STAINABILITY WITH BASE-SPECIFIC FLUOROCHROMES - DEPENDENCE ON THEDNA TOPOLOGY IN-SITU

The influence of DNA topology on stainability with the externally bind ing fluorochromes Hoechst 33258 (HO) and mithramycin (MI) was investig ated in HeLa nuclei in comparison with the intercalating dye propidium iodide (PI). Changes in DNA topology were induced with a mild DNAse I treatment. Stainability properties of untreated and nuclease-treated nuclei were compared with those of the supercoiled-circular and the re laxed-linear forms of the plasmid pBR322. DNAse-treated nuclei stained with HO showed a higher fluorescence intensity than control samples, independently of the dye concentration, in contrast with the findings obtained with PI. Similar behaviour was observed with the relaxed-line ar form of pBR322, compared with the supercoiled-circular molecule. Wi th MI, the stainability of HeLa nuclei did not depend on the DNA topol ogy, whereas the stainability of the plasmid was similar to that of HO , In order to assess whether this discrepancy depended on differences in the availability of DNAse-sensitive sites to the fluorochromes, flu orescence resonance energy transfer (FRET) studies were performed in n uclei stained with HO+PI, or with HO+MI dye pairs. After DNAse I diges tion, the relative FRET efficiency between donor (HO) and acceptor mol ecules (PI or MI) was reduced significantly only when MI was the accep tor. This result may be due to greater stainability of DNAse-sensitive sites with HO than with MI. These findings indicate that DNA stainabi lity with base-specific fluorochromes may be affected by the topology of chromatin regions.