In situ factors affecting stability of the DNA helix in interphase nuclei and metaphase chromosomes

The data from earlier cytochemical studies, in which the metachromatic fluorochrome acridine orange (AO) was used to differentially stain single vs double-stranded DNA, suggested that DNA in situ in intact metaphase chromosomes or in condensed chromatin of G0 cells is more sensitive to denaturation, induced by heat or acid, than DNA in decondensed chromatin of interphase nuclei. Present studies show that, indeed, DNA in permeabilized metaphase cells, in contrast to cells in interphase, when exposed to buffers of low pH (1.5-2.8) becomes digestible with the single-strand-specific S1 or mung bean nucleases. A variety of extraction procedures and enzymatic treatments provided evidence that the presence of histones, HMG proteins, and S-S bonds in chromatin, as well as phosphorylation or poly(ADP)ribosylation of chromatin proteins, can be excluded as a factor responsible for the differential sensitivity of metaphase vs interphase DNA to denaturation. Cell treatment with NaCl at a concentration of 1.2 N and above abolished the difference between interphase and mitotic cells, rendering DNA in mitotic cells less sensitive to denaturation; such treatment also resulted in decondensation of chromatin visible by microscopy. The present data indicate that structural proteins extractable with greater than or equal to 1.2 N NaCl may be involved in anchoring DNA to the nuclear matrix or chromosome scaffold and may be responsible for maintaining a high degree of chromatin compaction in situ, such as that observed in metaphase chromosomes or in G0 cells. Following dissociation of histones, the high spatial density of the charged DNA polymer may induce topological strain on the double helix, thus decreasing its local stability; this can be detected by metachromatic staining of DNA with AO or digestion with single-strand-specific nucleases.