Nick-forming sequences may be involved in the organization of eukaryotic chromatin into approximately 50 kbp loops.
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.
Székvölgyi
Lóránt
L
Department of Biophysics and Cell Biology, University Medical School of Debrecen, P.O. Box 39, 4012, Debrecen, Hungary.
Hegedüs
Eva
E
Molnár
Mónika
M
Bacsó
Zsolt
Z
Szarka
Krisztina
K
Beck
Zoltán
Z
Dombrádi
Viktor
V
Austin
Caroline
C
Szabó
Gábor
G
D017209:Apoptosis; D002453:Cell Cycle / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure; D002843:Chromatin / Q000648:ultrastructure*; D015825:Chromosomes, Fungal / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D053938:DNA Fragmentation; D004261:DNA Replication; D004587:Electrophoresis, Agar Gel; D005057:Eukaryotic Cells / Q000648:ultrastructure*; D005434:Flow Cytometry; D011495:Histone-Lysine N-Methyltransferase; D006801:Humans; D018613:Microscopy, Confocal; D051788:Myeloid-Lymphoid Leukemia Protein / Q000235:genetics; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000737:chemistry
D002843:Chromatin; C497422:KMT2A protein, human; D051788:Myeloid-Lymphoid Leukemia Protein; D004247:DNA; D011495:Histone-Lysine N-Methyltransferase; D015719:Single-Strand Specific DNA and RNA Endonucleases