PMID- 8831556
OWN - NLM
STAT- MEDLINE
DCOM- 19961105
LR  - 20131121
IS  - 0014-4827 (Print)
IS  - 0014-4827 (Linking)
VI  - 227
IP  - 2
DP  - 1996 Sep 15
TI  - DNA segments sensitive to single-strand-specific nucleases are present in 
      chromatin of mitotic cells.
PG  - 197-202
AB  - 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-stranded (ds) vs single-stranded (ss) nucleic 
      acids, as a marker of the degree of DNA denaturation. However, without prior cell 
      treatment with heat or acid no presence of single-stranded DNA in either mitotic 
      or interphase cells was detected by this assay. In the present experiments we 
      demonstrate that DNA in situ in mitotic cells, without any prior treatment that 
      can induce DNA denaturation, is sensitive to ss-specific S1 and mung bean 
      nucleases. Incubation of permeabilized human T cell leukemic MOLT-4, 
      promyelocytic HL-60, histiomonocytic lymphoma U937 cells, or normal 
      PHA-stimulated lymphocytes with S1 or mung bean nucleases 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 nucleases, DNA breakage in interphase cells was of an 
      order of magnitude less extensive compared to mitotic cells. The data indicate 
      that segments of DNA in mitotic chromosomes, in contrast to interphase cells, may 
      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 mitotic loops may lead to formation 
      of the hairpin-loop structures by inverted repeats; such structures are sensitive 
      to ss nucleases. The present method of detection of such segments appears to be 
      more sensitive than the use of AO. The identification of mitotic cells based on 
      sensitivity of their DNA to ss nucleases provides an additional method for their 
      quantification by flow cytometry.
FAU - Juan, G
AU  - Juan G
AD  - Cancer Research Institute, New York Medical College, Valhalla 10595, USA.
FAU - Pan, W
AU  - Pan W
FAU - Darzynkiewicz, Z
AU  - Darzynkiewicz Z
LA  - eng
GR  - R0 28704/PHS HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Exp Cell Res
JT  - Experimental cell research
JID - 0373226
RN  - 0 (Chromatin)
RN  - 0 (DNA, Single-Stranded)
RN  - 0 (Fluorescent Dyes)
RN  - EC 3.1.30.1 (Single-Strand Specific DNA and RNA Endonucleases)
RN  - G34N38R2N1 (Bromodeoxyuridine)
RN  - UT0S826Z60 (Uridine Triphosphate)
SB  - IM
MH  - Bromodeoxyuridine
MH  - Chromatin/*enzymology
MH  - DNA Fragmentation/physiology
MH  - DNA, Single-Stranded/*metabolism
MH  - Flow Cytometry
MH  - Fluorescent Dyes
MH  - Humans
MH  - Interphase/physiology
MH  - Leukemia, Promyelocytic, Acute
MH  - Lymphocytes/enzymology
MH  - Mitosis/*physiology
MH  - Nucleic Acid Denaturation
MH  - Single-Strand Specific DNA and RNA Endonucleases/*metabolism
MH  - Tumor Cells, Cultured/enzymology
MH  - Uridine Triphosphate/metabolism
EDAT- 1996/09/15 00:00
MHDA- 1996/09/15 00:01
CRDT- 1996/09/15 00:00
PHST- 1996/09/15 00:00 [pubmed]
PHST- 1996/09/15 00:01 [medline]
PHST- 1996/09/15 00:00 [entrez]
AID - S0014-4827(96)90267-0 [pii]
AID - 10.1006/excr.1996.0267 [doi]
PST - ppublish
SO  - Exp Cell Res. 1996 Sep 15;227(2):197-202. doi: 10.1006/excr.1996.0267.