title: Ribonucleoprotein-masked nicks at 50-kbp intervals in the eukaryotic genomic DNA.
abstract: 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.
authors.affiliation: Department of Biophysics and Cell Biology, Hungarian Academy of Sciences, Debrecen, Hungary.
authors.forename: Lóránt
authors.initials: L
authors.lastname: Székvölgyi
authors.forename: Zsuzsa
authors.initials: Z
authors.lastname: Rákosy
authors.forename: Bálint L
authors.initials: BL
authors.lastname: Bálint
authors.forename: Endre
authors.initials: E
authors.lastname: Kókai
authors.forename: László
authors.initials: L
authors.lastname: Imre
authors.forename: György
authors.initials: G
authors.lastname: Vereb
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Bacsó
authors.forename: Katalin
authors.initials: K
authors.lastname: Goda
authors.forename: Sándor
authors.initials: S
authors.lastname: Varga
authors.forename: Margit
authors.initials: M
authors.lastname: Balázs
authors.forename: Viktor
authors.initials: V
authors.lastname: Dombrádi
authors.forename: László
authors.initials: L
authors.lastname: Nagy
authors.forename: Gábor
authors.initials: G
authors.lastname: Szabó
mesh_terms: 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
chemical_list: D002843:Chromatin; D012261:Ribonucleoproteins; D051788:Myeloid-Lymphoid Leukemia Protein; D012313:RNA; D004247:DNA