RNA-DNA Hybrid Research Articles

DNA-RNA Hybrid (R-Loop): From a Unified Picture of the Mammalian Telomere to the Genome-Wide Profile

Local three-stranded DNA/RNA hybrid regions of genomes (R-loops) have been detected either by binding of a monoclonal antibody (DRIP assay) or by enzymatic recognition by RNaseH. Such a structure has been postulated for mouse and human telomeres, clearly suggested by the identification of the complementary RNA Telomeric repeat-containing RNA “TERRA”. However, the tremendous disparity in the information obtained with antibody-based technology drove us to investigate a new strategy. Based on the observation that DNA/RNA hybrids in a triplex complex genome co-purify with the double-stranded chromosomal DNA fraction, we developed a direct preparative approach from total protein-free cellular extract without antibody that allows their physical isolation and determination of their RNA nucleotide sequence. We then define in the normal mouse and human sperm genomes the notion of stable DNA associated RNA terminal R-loop complexes, including TERRA molecules synthesized from local promoters of every chromosome. Furthermore, the first strong evidence of all telomeric structures, applied additionally to the whole murine sperm genome compared to the testes, showed reproducible R-loop complexes of the whole genome and suggesting a defined profile in the sperm genome for the next generation.

Rapid and highly sensitive hairpin structure-mediated colorimetric detection of miRNA

Herein, we report a novel hairpin structure–mediated diagnostic method for the simple and rapid colorimetric detection of miRNA through the sensing of pyrophosphate. When the hairpin structure of the template DNA (h-Probe) was hybridized with the primer, the DNA primer extension mediated by nPfu special enzyme was blocked. However, this h-Probe was extended using nPfu special enzyme, upon the structural change of the template DNA, from a hairpin to a linear structure, in the presence of the target miRNA. The miRNA-hybridized template DNA sequence was cleaved by a duplex-specific nuclease (DSN), which cleaved the DNA from the RNA–DNA hybrid, thereby allowing the target miRNA to be recycled. Primer extension using nPfu special enzyme produced pyrophosphate when nucleotide triphosphate was incorporated into the DNA; this pyrophosphate was sensed in terms of a color change, from pink to colorless, when using pp Probe, a probe developed previously by our group. This novel system for the colorimetric detection of target miRNA operated with high sensitivity (LOD = 132 aM) and selectivity, with the whole detection process requiring only 30 min. Furthermore, this system could also detect miRNA fluorimetrically with similar sensitivity (LOD = 105 aM), highlighting the dual-sensing properties of pp Probe. This unique, extremely simple, and rapid system for the detection of miRNA through a highly sensitive color change would presumably be useful in applications requiring point-of-care detection.

Topoisomerase I Essentiality, DnaA-Independent Chromosomal Replication, and Transcription-Replication Conflict in Escherichia coli.

Topoisomerase I (Topo I) of Escherichia coli, encoded by topA, acts to relax negative supercoils in DNA. Topo I deficiency results in hypernegative supercoiling, formation of transcription-associated RNA-DNA hybrids (R-loops), and DnaA- and oriC-independent constitutive stable DNA replication (cSDR), but some uncertainty persists as to whether topA is essential for viability in E. coli and related enterobacteria. Here, we show that several topA alleles, including ΔtopA, confer lethality in derivatives of wild-type E. coli strain MG1655. Viability in the absence of Topo I was restored with two perturbations, neither of which reversed the hypernegative supercoiling phenotype: (i) in a reduced-genome strain (MDS42) or (ii) by an RNA polymerase (RNAP) mutation, rpoB*35, that has been reported to alleviate the deleterious consequences of RNAP backtracking and transcription-replication conflicts. Four phenotypes related to cSDR were identified for topA mutants: (i) one of the topA alleles rescued ΔdnaA lethality; (ii) in dnaA+ derivatives, Topo I deficiency generated a characteristic copy number peak in the terminus region of the chromosome; (iii) topA was synthetically lethal with rnhA (encoding RNase HI, whose deficiency also confers cSDR); and (iv) topA rnhA synthetic lethality was itself rescued by ΔdnaA. We propose that the terminal lethal consequence of hypernegative DNA supercoiling in E. coli topA mutants is RNAP backtracking during transcription elongation and associated R-loop formation, which in turn leads to transcription-replication conflicts and to cSDR. IMPORTANCE In all life forms, double-helical DNA exists in a topologically supercoiled state. The enzymes DNA gyrase and topoisomerase I act, respectively, to introduce and to relax negative DNA supercoils in Escherichia coli. That gyrase deficiency leads to bacterial death is well established, but the essentiality of topoisomerase I for viability has been less certain. This study confirms that topoisomerase I is essential for E. coli viability and suggests that in its absence, aberrant chromosomal DNA replication and excessive transcription-replication conflicts occur that are responsible for lethality.

A DNA packaging motor inchworms along one strand allowing it to adapt to alternative double-helical structures

Ring ATPases that translocate disordered polymers possess lock-washer architectures that they impose on their substrates during transport via a hand-over-hand mechanism. Here, we investigate the operation of ring motors that transport ordered, helical substrates, such as the bacteriophage ϕ29 dsDNA packaging motor. This pentameric motor alternates between an ATP loading dwell and a hydrolysis burst wherein it packages one turn of DNA in four steps. When challenged with DNA-RNA hybrids and dsRNA, the motor matches its burst to the shorter helical pitches, keeping three power strokes invariant while shortening the fourth. Intermittently, the motor loses grip on the RNA-containing substrates, indicating that it makes optimal load-bearing contacts with dsDNA. To rationalize these observations, we propose a helical inchworm translocation mechanism in which, during each cycle, the motor increasingly adopts a lock-washer structure during the ATP loading dwell and successively regains its planar form with each power stroke during the burst.

R-Loop-Mediated ssDNA Breaks Accumulate Following Short-Term Exposure to the HDAC Inhibitor Romidepsin.

Histone deacetylase inhibitors (HDACi) induce hyperacetylation of histones by blocking HDAC catalytic sites. Despite regulatory approvals in hematological malignancies, limited solid tumor clinical activity has constrained their potential, arguing for better understanding of mechanisms of action (MOA). Multiple activities of HDACis have been demonstrated, dependent on cell context, beyond the canonical induction of gene expression. Here, using a clinically relevant exposure duration, we established DNA damage as the dominant signature using the NCI-60 cell line database and then focused on the mechanism by which hyperacetylation induces DNA damage. We identified accumulation of DNA-RNA hybrids (R-loops) following romidepsin-induced histone hyperacetylation, with single-stranded DNA (ssDNA) breaks detected by single-cell electrophoresis. Our data suggest that transcription-coupled base excision repair (BER) is involved in resolving ssDNA breaks that, when overwhelmed, evolve to lethal dsDNA breaks. We show that inhibition of BER proteins such as PARP will increase dsDNA breaks in this context. These studies establish accumulation of R-loops as a consequence of romidepsin-mediated histone hyperacetylation. We believe that the insights provided will inform design of more effective combination therapy with HDACis for treatment of solid tumors. IMPLICATIONS: Key HDAC inhibitor mechanisms of action remain unknown; we identify accumulation of DNA-RNA hybrids (R-loops) due to chromatin hyperacetylation that provokes single-stranded DNA damage as a first step toward cell death.

P300/CBP Sustains Polycomb Silencing by Non-Enzymatic Functions

Maintenance of appropriate cell states involves epigenetic mechanisms, including Polycomb group (PcG)-mediated transcriptional repression. While PcG proteins are known to induce chromatin compaction, how PcG proteins gain access to DNA in compact chromatin to achieve long-term silencing is poorly understood. Here we show that the p300/CBP co-activator is associated with repressive chromatin regions and required for PcG occupancy in Drosophila and mouse cells. CBP stabilizes RNA polymerase II (Pol II) at PcG bound sites and promotes Pol II pausing independently of its histone acetyltransferase activity. CBP and Pol II pausing are necessary for RNA-DNA hybrid (R-loop) formation and nucleosome depletion at Polycomb Response Elements (PREs), whereas transcription beyond the pause region is not. These results suggest that non-enzymatic activities of the CBP co-activator have been repurposed to support PcG-mediated silencing, revealing how chromatin regulator interplay maintains transcriptional states.

RNA transcripts stimulate homologous recombination by forming DR-loops.

Homologous recombination (HR) repairs DNA double-strand breaks (DSBs) in the S and G2 phases of the cell cycle1-3. Several HR proteins are preferentially recruited to DSBs at transcriptionally active loci4-10, but how transcription promotes HR is poorly understood. Here we develop an assay to assess the effect of local transcription on HR. Using this assay, we find that transcription stimulates HR to a substantial extent. Tethering RNA transcripts to the vicinity of DSBs recapitulates the effects of local transcription, which suggests that transcription enhances HR through RNA transcripts. Tethered RNA transcripts stimulate HR in a sequence- and orientation-dependent manner, indicating that they function by forming DNA-RNA hybrids. In contrast to most HR proteins, RAD51-associated protein1 (RAD51AP1) only promotes HR when local transcription is active. RAD51AP1 drives the formation of R-loops in vitro and is required for tethered RNAs to stimulate HR in cells. Notably, RAD51AP1 is necessary for the DSB-induced formation of DNA-RNA hybrids in donor DNA, linking R-loops to D-loops. In vitro, RAD51AP1-generated R-loops enhance the RAD51-mediated formation of D-loops locally and give rise to intermediates that we term 'DR-loops', which contain both DNA-DNA and DNA-RNA hybrids and favour RAD51 function. Thus, at DSBs in transcribed regions, RAD51AP1 promotes the invasion of RNA transcripts into donor DNA, and stimulates HR through the formation of DR-loops.

PARP1-mediated PARylation of TonEBP prevents R-loop–associated DNA damage

Lack of coordination between the DNA replication and transcription machineries can increase the frequency of transcription–replication conflicts, leading ultimately to DNA damage and genomic instability. A major source of these conflicts is the formation of R-loops, which consist of a transcriptionally generated RNA–DNA hybrid and the displaced single-stranded DNA. R-loops play important physiological roles and have been implicated in human diseases. Although these structures have been extensively studied, many aspects of R-loop biology and R-loop–mediated genome instability remain unclear. We found that in cancer cells, tonicity-responsive enhancer-binding protein (TonEBP, also called NFAT5) interacted with PARP1 and localized to R-loops in response to DNA-damaging agent camptothecin (CPT), which is associated with R-loop formation. PARP1-mediated PARylation was required for recruitment of TonEBP to the sites of R-loop–associated DNA damage. Loss of TonEBP increased levels of R-loop accumulation and DNA damage, and promoted cell death in response to CPT. These findings suggest that TonEBP mediates resistance to CPT-induced cell death by preventing R-loop accumulation in cancer cells.

Mapping RNA with Nuclease S1

This protocol provides details for nuclease S1 mapping of mRNA using a uniformly labeled, single-stranded DNA probe. DNA-RNA hybrids are generated, which are subsequently digested with nuclease S1. The digestion products are separated using gel electrophoresis and analyzed by radiography.

Bacillus subtilis PcrA Helicase Removes Trafficking Barriers.

Bacillus subtilis PcrA interacts with the RNA polymerase and might contribute to mitigate replication–transcription conflicts (RTCs). We show that PcrA depletion lethality is partially suppressed by rnhB inactivation, but cell viability is significantly reduced by rnhC or dinG inactivation. Following PcrA depletion, cells lacking RnhC or DinG are extremely sensitive to DNA damage. Chromosome segregation is not further impaired by rnhB or dinG inactivation but is blocked by rnhC or recA inactivation upon PcrA depletion. Despite our efforts, we could not construct a ΔrnhC ΔrecA strain. These observations support the idea that PcrA dismantles RTCs. Purified PcrA, which binds single-stranded (ss) DNA over RNA, is a ssDNA-dependent ATPase and preferentially unwinds DNA in a 3′→5′direction. PcrA unwinds a 3′-tailed RNA of an RNA-DNA hybrid significantly faster than that of a DNA substrate. Our results suggest that a replicative stress, caused by mis-incorporated rNMPs, indirectly increases cell viability upon PcrA depletion. We propose that PcrA, in concert with RnhC or DinG, contributes to removing spontaneous or enzyme-driven R-loops, to counteract deleterious trafficking conflicts and preserve to genomic integrity.

Targeting the ALS/FTD-associated A-DNA kink with anthracene-based metal complex causes DNA backbone straightening and groove contraction.

The use of a small molecule compound to reduce toxic repeat RNA transcripts or their translated aberrant proteins to target repeat-expanded RNA/DNA with a G4C2 motif is a promising strategy to treat C9orf72-linked disorders. In this study, the crystal structures of DNA and RNA–DNA hybrid duplexes with the -GGGCCG- region as a G4C2 repeat motif were solved. Unusual groove widening and sharper bending of the G4C2 DNA duplex A-DNA conformation with B-form characteristics inside was observed. The G4C2 RNA–DNA hybrid duplex adopts a more typical rigid A form structure. Detailed structural analysis revealed that the G4C2 repeat motif of the DNA duplex exhibits a hydration shell and greater flexibility and serves as a ‘hot-spot’ for binding of the anthracene-based nickel complex, NiII(Chro)2 (Chro = Chromomycin A3). In addition to the original GGCC recognition site, NiII(Chro)2 has extended specificity and binds the flanked G:C base pairs of the GGCC core, resulting in minor groove contraction and straightening of the DNA backbone. We have also shown that Chro-metal complexes inhibit neuronal toxicity and suppresses locomotor deficits in a Drosophila model of C9orf72-associated ALS. The approach represents a new direction for drug discovery against ALS and FTD diseases by targeting G4C2 repeat motif DNA.

The rDNA is biomolecular condensate formed by polymer-polymer phase separation and is sequestered in the nucleolus by transcription and R-loops.

The nucleolus is the site of ribosome biosynthesis encompassing the ribosomal DNA (rDNA) locus in a phase separated state within the nucleus. In budding yeast, we find the rDNA locus and Cdc14, a protein phosphatase that co-localizes with the rDNA, behave like a condensate formed by polymer–polymer phase separation, while ribonucleoproteins behave like a condensate formed by liquid-liquid phase separation. The compaction of the rDNA and Cdc14’s nucleolar distribution are dependent on the concentration of DNA cross-linkers. In contrast, ribonucleoprotein nucleolar distribution is independent of the concentration of DNA cross-linkers and resembles droplets in vivo upon replacement of the endogenous rDNA locus with high-copy plasmids. When ribosomal RNA is transcribed from the plasmids by Pol II, the rDNA–binding proteins and ribonucleoprotein signals are weakly correlated, but upon repression of transcription, ribonucleoproteins form a single, stable droplet that excludes rDNA-binding proteins from its center. Degradation of RNA–DNA hybrid structures, known as R-loops, by overexpression of RNase H1 results in the physical exclusion of the rDNA locus from the nucleolar center. Thus, the rDNA locus is a polymer–polymer phase separated condensate that relies on transcription and physical contact with RNA transcripts to remain encapsulated within the nucleolus.

DNA Breaks-Mediated Fitness Cost Reveals RNase HI as a New Target for Selectively Eliminating Antibiotic-Resistant Bacteria.

Antibiotic resistance often generates defects in bacterial growth called fitness cost. Understanding the causes of this cost is of paramount importance, as it is one of the main determinants of the prevalence of resistances upon reducing antibiotics use. Here we show that the fitness costs of antibiotic resistance mutations that affect transcription and translation in Escherichia coli strongly correlate with DNA breaks, which are generated via transcription–translation uncoupling, increased formation of RNA–DNA hybrids (R-loops), and elevated replication–transcription conflicts. We also demonstrated that the mechanisms generating DNA breaks are repeatedly targeted by compensatory evolution, and that DNA breaks and the cost of resistance can be increased by targeting the RNase HI, which specifically degrades R-loops. We further show that the DNA damage and thus the fitness cost caused by lack of RNase HI function drive resistant clones to extinction in populations with high initial frequency of resistance, both in laboratory conditions and in a mouse model of gut colonization. Thus, RNase HI provides a target specific against resistant bacteria, which we validate using a repurposed drug. In summary, we revealed key mechanisms underlying the fitness cost of antibiotic resistance mutations that can be exploited to specifically eliminate resistant bacteria.

A triskelion of nucleic acids drives protein aggregation in A-T

Mutations in ataxia telangiectasia mutated (ATM) kinase lead to cerebellar neurodegeneration. In this issue of Molecular Cell, Lee etal. (2021) revealed how transcription-induced reactive oxygen species and DNA-RNA hybrids activate PARP enzymes, generating the nucleic acid poly-ADP-ribose, which promotes the accumulation of protein aggregates in A-T-like disorders.

An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing

Extensive testing is essential to break the transmission of SARS-CoV-2, which causes the ongoing COVID-19 pandemic. Here, we present a CRISPR-based diagnostic assay that is robust to viral genome mutations and temperature, produces results fast, can be applied directly on nasopharyngeal (NP) specimens without RNA purification, and incorporates a human internal control within the same reaction. Specifically, we show that the use of an engineered AsCas12a enzyme enables detection of wildtype and mutated SARS-CoV-2 and allows us to perform the detection step with loop-mediated isothermal amplification (LAMP) at 60-65 °C. We also find that the use of hybrid DNA-RNA guides increases the rate of reaction, enabling our test to be completed within 30 minutes. Utilizing clinical samples from 72 patients with COVID-19 infection and 57 healthy individuals, we demonstrate that our test exhibits a specificity and positive predictive value of 100% with a sensitivity of 50 and 1000 copies per reaction (or 2 and 40 copies per microliter) for purified RNA samples and unpurified NP specimens respectively.

Gene Co-Expression Analysis of Human RNASEH2A Reveals Functional Networks Associated with DNA Replication, DNA Damage Response, and Cell Cycle Regulation.

Simple SummaryRNASEH2A is the catalytic subunit of the ribonuclease (RNase) H2 ternary complex that plays an important role in maintaining DNA stability in cells. Recent studies have shown that the RNASEH2A subunit alone is highly expressed in certain cancer cell types. Via a series of bioinformatics approaches, we found that RNASEH2A is highly expressed in human proliferative tissues and many cancers. Our analyses reveal a possible involvement of RNASEH2A in cell cycle regulation in addition to its well established role in DNA replication and DNA repair. Our findings underscore that RNASEH2A could serve as a biomarker for cancer diagnosis and a therapeutic target.Ribonuclease (RNase) H2 is a key enzyme for the removal of RNA found in DNA-RNA hybrids, playing a fundamental role in biological processes such as DNA replication, telomere maintenance, and DNA damage repair. RNase H2 is a trimer composed of three subunits, RNASEH2A being the catalytic subunit. RNASEH2A expression levels have been shown to be upregulated in transformed and cancer cells. In this study, we used a bioinformatics approach to identify RNASEH2A co-expressed genes in different human tissues to underscore biological processes associated with RNASEH2A expression. Our analysis shows functional networks for RNASEH2A involvement such as DNA replication and DNA damage response and a novel putative functional network of cell cycle regulation. Further bioinformatics investigation showed increased gene expression in different types of actively cycling cells and tissues, particularly in several cancers, supporting a biological role for RNASEH2A but not for the other two subunits of RNase H2 in cell proliferation. Mass spectrometry analysis of RNASEH2A-bound proteins identified players functioning in cell cycle regulation. Additional bioinformatic analysis showed that RNASEH2A correlates with cancer progression and cell cycle related genes in Cancer Cell Line Encyclopedia (CCLE) and The Cancer Genome Atlas (TCGA) Pan Cancer datasets and supported our mass spectrometry findings.

CENP-A chromatin prevents replication stress at centromeres to avoid structural aneuploidy

Chromosome segregation relies on centromeres, yet their repetitive DNA is often prone to aberrant rearrangements under pathological conditions. Factors that maintain centromere integrity to prevent centromere-associated chromosome translocations are unknown. Here, we demonstrate the importance of the centromere-specific histone H3 variant CENP-A in safeguarding DNA replication of alpha-satellite repeats to prevent structural aneuploidy. Rapid removal of CENP-A in S phase, but not other cell-cycle stages, caused accumulation of R loops with increased centromeric transcripts, and interfered with replication fork progression. Replication without CENP-A causes recombination at alpha-satellites in an R loop-dependent manner, unfinished replication, and anaphase bridges. In turn, chromosome breakage and translocations arise specifically at centromeric regions. Our findings provide insights into how specialized centromeric chromatin maintains the integrity of transcribed noncoding repetitive DNA during S phase.

BRCA2 promotes DNA-RNA hybrid resolution by DDX5 helicase at DNA breaks to facilitate their repair‡.

The BRCA2 tumor suppressor is a DNA double-strand break (DSB) repair factor essential for maintaining genome integrity. BRCA2-deficient cells spontaneously accumulate DNA-RNA hybrids, a known source of genome instability. However, the specific role of BRCA2 on these structures remains poorly understood. Here we identified the DEAD-box RNA helicase DDX5 as a BRCA2-interacting protein. DDX5 associates with DNA-RNA hybrids that form in the vicinity of DSBs, and this association is enhanced by BRCA2. Notably, BRCA2 stimulates the DNA-RNA hybrid-unwinding activity of DDX5 helicase. An impaired BRCA2-DDX5 interaction, as observed in cells expressing the breast cancer variant BRCA2-T207A, reduces the association of DDX5 with DNA-RNA hybrids, decreases the number of RPA foci, and alters the kinetics of appearance of RAD51 foci upon irradiation. Our findings are consistent with DNA-RNA hybrids constituting an impediment for the repair of DSBs by homologous recombination and reveal BRCA2 and DDX5 as active players in their removal.

RNA polymerase III is required for the repair of DNA double-strand breaks by homologous recombination

End resection in homologous recombination (HR) and HR-mediated repair of DNA double-strand breaks (DSBs) removes several kilobases from 5' strands of DSBs, but 3' strands are exempted from degradation. The mechanism by which the 3' overhangs are protected has not been determined. Here, we established that the protection of 3' overhangs is achieved through the transient formation of RNA-DNA hybrids. The DNA strand in the hybrids is the 3' ssDNA overhang, while the RNA strand is newly synthesized. RNA polymerase III (RNAPIII) is responsible for synthesizing the RNA strand. Furthermore, RNAPIII is actively recruited to DSBs by the MRN complex. CtIP and MRN nuclease activity is required for initiating the RNAPIII-mediated RNA synthesis at DSBs. A reduced level of RNAPIII suppressed HR, and genetic loss > 30bp increased at DSBs. Thus, RNAPIII is an essential HR factor, and the RNA-DNA hybrid is an essential repair intermediate for protecting the 3' overhangs in DSB repair.

A simple screening strategy for complex RNA-DNA hybrid nanoshapes.

The design of hybrid nucleic acid nanomaterials capitalizes on the partitioning of architectural and functional roles between structurally diverse RNA modules and chemically robust DNA components. Selecting optimal combinations of RNA and DNA building blocks is the key to preparing stable polygonal RNA-DNA hybrid nanoshapes. Here, we outline a simple screening strategy by gel electrophoresis under native folding conditions to identify combinations of RNA and DNA modules that self-assemble to robust polygonal hybrid nanoshapes. As a proof of concept, we outline the preparation of RNA-DNA hybrid nanoshapes containing a set of different RNA architectural joints, including internal loop motifs and three-way junction (3WJ) folds. For each hybrid nanoshape, we demonstrate the selection process used to identify optimal DNA modules from a library of DNA connectors. The simple screening strategy outlined here provides a general robust method to identify and prepare RNA-DNA hybrid nanoshapes from diverse libraries of discrete nucleic acid building blocks.