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

Abstract

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.

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

“…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.…”

Separation of 1–23-kb complementary DNA strands by urea–agarose gel electrophoresis

“…The Saccharomyces cerevisiae WDHY 199 (MATa, leu2-3,112 trp1-289 ura3-52 his7-2 lys1-1) cells were grown and the preparation of agarose-plugs containing the yeast spheroplasts was carried out as previously described (16). For restriction enzyme digestion, the plugs were preincubated in the appropriate 1× restriction enzyme buffers three times for 1 h each, then incubated with 150 U/ml SmaI or Nb.Bpu10I (Fermentas Life Science, Maryland, USA) in 200 μl of the same buffer at 37°C, for 1.5 h. For S1 nuclease treatment, 1× S1 buffer was used for washing of the plugs before digestion by 500 U/ml of the enzyme (Promega Life Science, Madison, USA) at 37°C, for 1.5 h. The plugs were finally equilibrated with TE buffer before electrophoresis.…”

“…PCR was performed using 1.25 U of the Long PCR Enzyme mix (Fermentas) in 50 μl of 1× buffer supplemented with 1.5 mM MgCl 2 , containing, 20 pmol of each primer (Integrated DNA Technologies, Coralville, IA, USA), the dNTPs (Promega) at 0.25 mM concentration and 300 ng S. cerevisae genomic DNA prepared as described earlier (16). Each forward primer (see Tables 1–2 of Supplementary data) was used in pair with the reverse p1R primer resulting in variable length of amplicons, overlapping at their 3′-ends defined by the common reverse primer.…”

The histochemistry and cell biology vade mecum: a review of 2005–2006

“…The existence of special, hypersensitive regions positioned regularly at approximately 50 kbp loop-size intervals in the eukaryotic chromatin was investigated by Szekvolgyi et al (2006). In yeast cell systems, they found that loop-size fragmentation can occur in any phase of the cell cycle.…”