Single-Strand Breaks in Agarose-Embedded Chromatin of Nonapoptotic Cells

Varga, Tamás; Szilágyi, I; Szabó, Gábor

doi:10.1006/bbrc.1999.1470

Cited by 10 publications

(22 citation statements)

References 39 publications

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“…Thus, the en masse chromatin fragmentation observed is definitely not due to the presence of dead or apoptosizing cells. These data fully confirm and extend our previous observation on stationary phase cells of a different strain of S. cerevisiae (Varga et al 1999) and parallel our similar findings on normal, healthy mammalian cells (Gal et al 2000;Szabo 1995;Szabo et al 1987Szabo et al , 1990Szilagyi et al 2003). Since DNA runs slower when denatured, accounting for the different migration of S1-digested and denatured fragments (data not shown), the two phenomena of chromatin fragmentation are suggested to occur at the same or spatially juxtaposed sites.…”

Section: Discussionsupporting

confidence: 92%

“…Furthermore, a similar fragmentation is observed upon S1 treatment of the deproteinized samples. Since both the above phenomena have been observed in several different mammalian and rodent cell lines (Szabo 1995;Szabo et al 1990), as well as in several yeast strains (Varga et al 1999 and data herein), loop-size chromatin fragmentation upon deproteinization appears to be a general feature of eukaryotic chromatin.…”

Section: Discussionmentioning

confidence: 55%

“…loop-size molecules (Gal et al 2000;Szabo 1995;Szabo et al 1987Szabo et al , 1990Szilagyi et al 2003;Varga et al 1999). The extent of proteolytic digestion appears to be crucial in determining the apparent size of purified DNA, with $50 kbp fragments yielded by complete digestion (Szabo 1995).…”

Section: Introductionmentioning

confidence: 99%

“…The extent of proteolytic digestion appears to be crucial in determining the apparent size of purified DNA, with $50 kbp fragments yielded by complete digestion (Szabo 1995). Although (a) the size range of these fragments is sometimes surprisingly narrow (Szabo et al1990), (b) handling artifacts are not involved in eliciting fragmentation (Szabo 1995;Varga et al 1999) and (c) it is observed without extensive proteolytic treatments in the case of apoptotic cells (Oberhammer et al 1993), mechanical factors are generally considered as its major cause. This interpretation is based on the fact that fragmentation is avoided if agarose-embedded cells are exposed to the lysing solutions (Vollrath and Davis 1987).…”

Section: Introductionmentioning

confidence: 99%

“…This interpretation is based on the fact that fragmentation is avoided if agarose-embedded cells are exposed to the lysing solutions (Vollrath and Davis 1987). However, the high molecular weight DNA obtained in this procedure is turned into loop-size fragments when the agarose plugs are melted up to 60-95°C or upon exposure to S1 nuclease, as shown for different mammalian cells as well as stationary phase yeast cultures (Varga et al 1999). These latter phenomena may be interpreted to suggest that singlestranded discontinuities, probably nicks, may be present in the lysed nuclei, and that these discontinuities are prevented from becoming manifest double-strand breaks when enclosed in an agarose matrix.…”

Section: Introductionmentioning

confidence: 99%

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Nick-forming sequences may be involved in the organization of eukaryotic chromatin into ∼50 kbp loops

Székvölgyi

Hegedüs

Molnár

et al. 2005

Histochem Cell Biol

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Phenomena involving the disassembly of chromosomes to approximately 50 kbp double-stranded fragments upon protein denaturing treatments of normal and apoptotic mammalian nuclei as well as yeast protoplasts may be an indication of special, hypersensitive regions positioned regularly at loop-size intervals in the eukaryotic chromatin. Here we show evidence in yeast cell systems that loop-size fragmentation can occur in any phase of the cell cycle and that the plating efficiency of these cells is approximately 100%. The possibility of sequence specificity was investigated within the breakpoint cluster region (bcr) of the human MLL gene, frequently rearranged in certain leukemias. Our data suggest that DNA isolated from yeast cultures or mammalian cell lines carry nicks or secondary structures predisposing DNA for a specific nicking activity, at non-random positions. Furthermore, exposure of MLL bcr-carrying plasmid DNA to S1 nuclease or nuclear extracts or purified topoisomerase II elicited cleavages at the nucleotide positions of nick formation on human genomic DNA. These data support the possibility that certain sequence elements are preferentially involved in the cleavage processes responsible for the en masse disassembly of chromatin to loop-size fragments upon isolation of DNA from live eukaryotic cells.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 55%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Nick-forming sequences may be involved in the organization of eukaryotic chromatin into ∼50 kbp loops

Székvölgyi

Hegedüs

Molnár

et al. 2005

Histochem Cell Biol

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Non‐random features of loop‐size chromatin fragmentation

Szilágyi

Varga

Székvölgyi

et al. 2003

J of Cellular Biochemistry

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Upon isolation of DNA from normal eukaryotic cells by standard methods involving extensive proteolytic treatment, a rather homogeneous population of loop-size, double-stranded DNA fragments is regularly obtained. These DNA molecules can be efficiently end-labeled by the DNA polymerase I Klenow fragment, as well as by a 3'- to -5'-exonuclease-free Klenow enzyme, but not by terminal transferase (TdT) unless the ends have been filled up by Klenow, suggesting that dominantly 5' protruding termini are generated upon fragmentation. The filled-up termini were used for cloning the distal parts of the approximately 50 kb fragments. BLAST analysis of the sequence of several clones allowed us to determine the sequence of the non-cloned side of the breakpoints. Comparison of 25, 600 bp-long breakpoint sequences demonstrated prevalence of repetitive elements. Consensus motives characteristic of the breakpoint sequences have been identified. Several sequences exhibit peculiar computed conformational characteristics, with sharp transition or center of symmetry located exactly at the breakpoint. Our data collectively suggest that chromatin fragmentation involves nucleolytic cleavages at fragile/hypersensitive sites delimiting loop-size fragments in a non-random manner. Interestingly, the sequence characteristics of the breakpoints are reminiscent of certain breakpoint cluster regions frequently subject to gene rearrangements.

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