Protease inducible alkali lability of DNA from proliferating and non-proliferating cells

Werner, Dieter; Hadjiolov, D.; Neuer, B.

doi:10.1016/0006-291x(81)91929-x

Cited by 9 publications

(5 citation statements)

References 5 publications

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“…Our findings are reminiscent of early observations by Weintraub (5), demonstrating the release of discrete supranucleosomal ''a'' particles containing Ϸ20-to 40-kbp dsDNA upon mild nuclease digestion of chromatin and of those by Werner et al (20)(21)(22) on the presence of protease-induced S1 nuclease-and alkalinesensitive regions arranged at Ϸ13.5-and 27-kbp intervals, respectively, in Ehrlich ascites tumor cell DNA. In the latter experiments, additional labile sites of different configuration may have been revealed (23), because the ends of those shorter DNA fragments, observed at more intensively denaturing conditions, were not labeled by Pol I. Alternatively, secondary changes might have been generated in that or in our system, leading to different end structures.…”

Section: Discussionsupporting

confidence: 73%

Ribonucleoprotein-masked nicks at 50-kbp intervals in the eukaryotic genomic DNA

Székvölgyi¹,

Rákosy²,

Bálint³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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By using a microscopic approach, field inversion single-cell gel electrophoresis, we show that preformed single-strand discontinuities are present in the chromatin of resting and proliferating mammalian and yeast cells. These single-strand breaks are primarily nicks positioned at Ϸ50-kbp intervals throughout the entire genome that could be efficiently labeled in situ by DNA polymerase I holoenzyme but not by Klenow fragment and terminal transferase unless after ribonucleolytic treatments. The RNA molecules involved appear to comprise R-loops, recognized by the S9.6 RNA/DNA hybrid-specific antibody. By using the breakpoint cluster region of the Mixed Lineage Leukemia (MLL) gene as a model, we have found that the number of manifest nicks detected by FISH performed after field inversion single-cell gel electrophoresis depends on epigenetic context, but the difference between germ-line and translocated MLL alleles is abolished by protease treatment. Our data imply that the double-stranded genomic DNA is composed of contiguous rather than continuous single strands and reveal an aspect of higher-order chromatin organization with ribonucleoprotein-associated persistent nicks defining Ϸ50-kbp domains.chromatin loop ͉ RNA/DNA hybrid ͉ translocation T he concept that eukaryotic chromatin is organized into Ϸ30-to 150-kbp units anchored to a ribonucleoprotein-containing structure, the enigmatic nuclear matrix/scaffold, has stemmed from microscopic observations of DNA loops emanating from histone-depleted nuclei (for review, see ref 1). Chromatin appears to bind matrix elements through special, although heterogeneous, DNA sequences, scaffold/matrix attachment regions (S/MARs), that remain attached to the remnants of saltextracted nuclei (nuclear halos) and are thought to represent the boundaries of supercoiled 20-to 150-kbp looped domains (2). Consistent with this model of chromosome architecture, chromatin fragmentation phenomena have been observed that involve the preferential cleavage of DNA, presumably at the bases of loops (3). The global disassembly of chromatin to highmolecular-weight (Ն20-kbp) units also takes place upon alkali denaturation after proteinase digestion (4), at exposure to single-strand (ss)-specific nuclease (5), in the early stage of apoptotic DNA fragmentation (6), as well as in the case of healthy nonapoptotic mammalian and yeast cells upon various protein denaturing treatments (7,8). The DNase I hypersensitivity of mammalian chromatin at every Ϸ50 kbp (9), also detected in the vicinity of certain S/MARs (10), points to the special vulnerability of the DNA at the borders of supernucleosomal units of this size. The above data raise the question whether special base-unpaired secondary structures or perhaps regularly spaced stably maintained ss discontinuities constitute the predilection points of Ϸ50-kbp chromatin fragmentation, delimiting higher-order domains. To tackle this issue, based on the conventional comet assay (11), we have developed a microscopic approach, field inversion single-cell gel el...

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Section: Discussionsupporting

confidence: 73%

Ribonucleoprotein-masked nicks at 50-kbp intervals in the eukaryotic genomic DNA

Székvölgyi¹,

Rákosy²,

Bálint³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…However, neither of the TBPs appears to be related to the 50-or 55-kd prekeratin proteins (lanes 2-5) or to 1-tubulin (lane 10). We therefore conclude that the TBPs reported here and those reported by Werner and his co-workers (7,28,54,(60)(61)(62)(63) (29,32,55) and silver stained (36). Lane 1; HeLa cell TBPs isolated by SDS-Sepharose 4B-CL column chromatography; lane 2, proteins associated with HeLa cell DNA after cell lysis in alkaline SDS and phenol extraction by the method of Werner et al (63).…”

Section: Resultssupporting

confidence: 68%

“…They are specifically enriched in the nuclear matrix fraction, even after 98% of the cellular DNA is removed by nuclease digestion and the size of the bulk DNA is reduced to approximately 150 base pairs (bp). These proteins also possess peptide map homology to proteins previously reported to be bound to a wide variety of procaryotic, eucaryotic, and plant cells DNAs, even after alkali treatment, pronase digestion, and phenol extraction (7,28,(60)(61)(62)(63). These observations strongly indicate that the HeLa cell proteins are involved in anchoring DNA on the nuclear matrix and that similar proteins participate in the organization of chromosomal domains in both procaryotic and eucaryotic cells.…”

supporting

confidence: 62%

“…However, for reasons enumerated below, we feel that the TBPs are generally underrepresented in the pool of nuclear matrix proteins. The molecular weights of the HeLa cell TBPs were the same as those reported by D. Werner and his co-workers for the proteins found tightly bound to DNA extracted from mammalian, plant, and bacterial cells (7,28,54,(60)(61)(62)(63). To determine whether these proteins were the same as the TBPs present in the nuclear-matrix fraction, we prepared DNA-protein complexes from HeLa cells by using the method of Werner et al (63).…”

Section: Resultsmentioning

confidence: 72%

“…Werner and associates (7,28,54,(60)(61)(62)(63) have shown that two proteins, reported as 54 and 68 kd, are found tightly bound to the DNA of many cell types (including those of mammals, plants, and bacteria), even after the DNA is treated with alkali, detergent, and proteases. Furthermore, Werner et al (62) have shown that antibodies raised to the TBPs from mouse cells will immunologically cross-react with the TBPs associated with E. coli DNA.…”

Section: Resultsmentioning

confidence: 99%

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Proteins tightly bound to HeLa cell DNA at nuclear matrix attachment sites.

Bodnar¹,

Jones²,

Coombs³

et al. 1983

Mol. Cell. Biol.

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DNA-protein complexes have been isolated from HeLa cell nuclei and nuclear matrix preparations. Two proteins, 55 and 66 kilodaltons in size, remain bound to HeLa DNA after treatment at 80°C in 2% sodium dodecyl sulfate and purification by exclusion chromatography on Sepharose 2B-CL in the presence of 0.3% sodium dodecyl sulfate. These proteins appear to be tightly bound but not covalently linked to the DNA, and they are distributed over the DNA with an average spacing of 40 kilobase pairs. This spacing distribution remains essentially constant throughout the cell cycle. The proteins are bound to the residual 2% of HeLa cell DNA which remains attached to the nuclear matrix after extensive nuclease digestion, a condition which reduces the average size of the DNA to -150 base pairs. Our results suggest that these tightly bound proteins are involved in anchoring cellular DNA to the nuclear matrix. These tightly bound proteins are identical by partial peptide mapping to proteins found tightly bound to the DNA of mammalian, plant, and bacterial cells (D. Werner and C. Petzelt, J. Mol. Biol. 150:297-302, 1981), implying that these proteins are involved in the organization of chromosomal domains and are highly conserved in both procaryotic and eucaryotic cells.The DNA of eucaryotic cells is organized into structural domains 30 to 90 kilobases (kb) in size. These domains are seen in metaphase chromosomes as loops or rosettes of DNA bound to a protein scaffold (10,40,43). In interphase nuclei the DNA is organized in domains of supercoiling measured at 84 to 94 kb (4,22,57), and the DNA is attached to the nuclear matrix with an average spacing of between 35 and 60 kb (25,48). Cellular DNA replication also takes place in association with the nuclear matrix (16,34,42,58), and eucaryotic DNA replicons have a size distribution similar to that of the structural domains described above (18). Several proteins that are tightly bound to cellular DNA have been implicated in maintaining supercoiled domains, binding DNA to chromosome and nuclear scaffolds, or binding to transcriptionally active genes (3,24,28,37,46,47 1981). However, proteins that mediate the interaction between cellular DNA and the nuclear matrix have not been defined.Using methods initially developed to study the adenovirus terminal protein-DNA complex (14, 21, 50), we isolated a stable DNA-protein complex from HeLa cells that contains two proteins, 55 and 66 kilodaltons (kd) in size. These proteins are tightly bound to cellular DNA and exhibit a constant average spacing of about 40 kb throughout the cell cycle. They are specifically enriched in the nuclear matrix fraction, even after 98% of the cellular DNA is removed by nuclease digestion and the size of the bulk DNA is reduced to approximately 150 base pairs (bp). These proteins also possess peptide map homology to proteins previously reported to be bound to a wide variety of procaryotic, eucaryotic, and plant cells DNAs, even after alkali treatment, pronase digestion, and phenol extraction (7,28,(60)(61)(62)(63...

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