Ribonucleoprotein-masked nicks at 50-kbp intervals in the eukaryotic genomic DNA.
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 approximately 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 approximately 50-kbp domains.
Székvölgyi
Lóránt
L
Department of Biophysics and Cell Biology, Hungarian Academy of Sciences, Debrecen, Hungary.
Rákosy
Zsuzsa
Z
Bálint
Bálint L
BL
Kókai
Endre
E
Imre
László
L
Vereb
György
G
Bacsó
Zsolt
Z
Goda
Katalin
K
Varga
Sándor
S
Balázs
Margit
M
Dombrádi
Viktor
V
Nagy
László
L
Szabó
Gábor
G
D049109:Cell Proliferation; D002478:Cells, Cultured; D002843:Chromatin / Q000648:ultrastructure; D020552:Comet Assay; D004247:DNA / Q000378:metabolism; D053904:DNA Breaks, Single-Stranded; D053938:DNA Fragmentation; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D019169:Jurkat Cells; D051788:Myeloid-Lymphoid Leukemia Protein / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
D002843:Chromatin; D012261:Ribonucleoproteins; D051788:Myeloid-Lymphoid Leukemia Protein; D012313:RNA; D004247:DNA