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Ribonucleoprotein-Masked Nicks at 50-kbp Intervals in the Eukaryotic Genomic DNA
Lóránt Székvölgyi, Zsuzsa Rákosy, Bálint L. Bálint, Endre Kókai, László Imre, György Vereb, Zsolt Bacsó, Katalin Goda, Sándor Varga, Margit Balázs, Viktor Dombrádi, László Nagy and Gábor Szabó
Proceedings of the National Academy of Sciences of the United States of America
Vol. 104, No. 38 (Sep. 18, 2007), pp. 14964-14969
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/25449067
Page Count: 6
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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 ≈50-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 ≈50-kbp domains.
Proceedings of the National Academy of Sciences of the United States of America © 2007 National Academy of Sciences