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 Ϸ50-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 Ϸ50-kbp domains.chromatin loop ͉ RNA/DNA hybrid ͉ translocation T he concept that eukaryotic chromatin is organized into Ϸ30-to 150-kbp units anchored to a ribonucleoprotein-containing structure, the enigmatic nuclear matrix/scaffold, has stemmed from microscopic observations of DNA loops emanating from histone-depleted nuclei (for review, see ref 1). Chromatin appears to bind matrix elements through special, although heterogeneous, DNA sequences, scaffold/matrix attachment regions (S/MARs), that remain attached to the remnants of saltextracted nuclei (nuclear halos) and are thought to represent the boundaries of supercoiled 20-to 150-kbp looped domains (2). 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. The above data raise the question whether special base-unpaired secondary structures or perhaps regularly spaced stably maintained ss discontinuities constitute the predilection points of Ϸ50-kbp chromatin fragmentation, delimiting higher-order domains. To tackle this issue, based on the conventional comet assay (11), we have developed a microscopic approach, field inversion single-cell gel el...

RNA-DNA hybrid (R-loop) immunoprecipitation mapping: an analytical workflow to evaluate inherent biases

“…The above examples clearly illustrate the massive progress in the field that has been driven by technological advancements of Rloop detection methods. These techniques involve, for instance, electrophoretic mobility shift assays (Yu et al 2006), atomic force microscopy (Brown et al 2008), transmission electron microscopy (Pohjoismäki et al 2010), fluorescent microscopy (Székvölgyi et al 2007), fluorescence in situ hybridization (Nadel et al 2015), native bisulfite modification (Yu et al 2003), immunoprecipitation (Skourti-Stathaki et al 2011;Ginno et al 2012), and computational prediction (Jenjaroenpun et al 2015). The increasing numbers of R-loop mapping data relied on a single approach, DNA-RNA immunoprecipitation (DRIP) and its variations (RDIP, DRIPc, S1-DRIP, DRIP-RNA, DIP, ChIP).…”

The monoclonal S9.6 antibody exhibits highly variable binding affinities towards different R-loop sequences

“…R-loops can now be effectively mapped with high-throughput methods that are based on the specific recognition of RNA-DNA hybrids by the S9.6 antibody [14,15]. The antibody was recently used to detect and localize DNA—RNA hybrids that have been linked to genomic instability, at CpG island promoters, terminator regions and genomic regions with altered chromatin structure [16–19] [9,20]. …”

A structural basis for cellular senescence

“…Interestingly, in these cells terminal differentiation is linked to induction of DNA strands breaks that preferentially occur at sites involving MARs, liberating DNA fragments of some 50 kbp, that roughly correspond to the average distribution of chromatin looped domains [88]. Indeed, ribo-nucleoprotein-masked nicks exist in the genome distributed on average every 50 kbp, suggesting that eukaryotic genomic DNA is composed of contiguous rather than continuous single strands, interrupted at the boundaries of interphase chromatin loops [89]. This fact supports the notion that attachment to the NM contributes to stabilize the long-range DNA structure.…”