DNA SEGMENTS SENSITIVE TO SINGLE-STRAND-SPECIFIC NUCLEASES ARE PRESENT IN CHROMATIN OF MITOTIC CELLS

It was observed before that DNA in situ in chromatin of mitotic cells is more sensitive to denaturation than DNA in chromatin of interphase cells. DNA sensitivity to denaturation, in these studies, was analyzed by exposing cells to heat or acid and using acridine orange (AO), the metachromatic fluorochrome which can differentially stain double-stra nded (ds) vs single-stranded (ss) nucleic acids, as a marker of the de gree of DNA denaturation. However, without prior cell treatment with h eat or acid no presence of single-stranded DNA in either mitotic or in terphase cells was detected by this assay. In the present experiments we demonstrate that DNA in situ in mitotic cells, without any prior tr eatment that can induce DNA denaturation, is sensitive to ss-specific S1 and mung bean nucleases. Incubation of permeabilized human T cell l eukemic MOLT-4, promyelocytic HL-60, histiomonocytic lymphoma U937 cel ls, or normal PHA-stimulated lymphocytes with S1 or mung bean nuclease s generated extensive DNA breakage in mitotic cells, DNA strand breaks were detected using fluorochrome-labeled triphosphonucleotides in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase . Under identical conditions of the cells' exposure to ss-specific nuc leases, DNA breakage in interphase cells was of an order of magnitude less extensive compared to mitotic cells. The data indicate that segme nts of DNA in mitotic chromosomes, in contrast to interphase cells, ma y be in a conformation which is sensitive to ss nucleases. This may be a reflection of the differences in the torsional stress of DNA loops between interphase and mitotic chromatin. Namely, greater stress in mi totic loops may lead to formation of the hairpin-loop structures by in verted repeats; such structures are sensitive to ss nucleases. The pre sent method of detection of such segments appears to be more sensitive than the use of AO. The identification of mitotic cells based on sens itivity of their DNA to ss nucleases provides an additional method for their quantification by flow cytometry. (C) 1996 Academic Press, Inc.