Formation Of RNA-DNA Hybrids Research Articles

RAD52 resolves transcription-replication conflicts to mitigate R-loop induced genome instability

Collisions of the transcription and replication machineries on the same DNA strand can pose a significant threat to genomic stability. These collisions occur in part due to the formation of RNA-DNA hybrids termed R-loops, in which a newly transcribed RNA molecule hybridizes with the DNA template strand. This study investigated the role of RAD52, a known DNA repair factor, in preventing collisions by directing R-loop formation and resolution. We show that RAD52 deficiency increases R-loop accumulation, exacerbating collisions and resulting in elevated DNA damage. Furthermore, RAD52’s ability to interact with the transcription machinery, coupled with its capacity to facilitate R-loop dissolution, highlights its role in preventing collisions. Lastly, we provide evidence of an increased mutational burden from double-strand breaks at conserved R-loop sites in human tumor samples, which is increased in tumors with low RAD52 expression. In summary, this study underscores the importance of RAD52 in orchestrating the balance between replication and transcription processes to prevent collisions and maintain genome stability.

Abstract 6214: FANCA promotes transcription-coupled homologous recombination by catalyzing R-loops formation

Abstract R loops arise from hybridization of RNA transcripts with template DNA during transcription, which are not only cause DNA damage in certain contexts as cancer hallmarks but also be important regulators of cellular processes. Recent findings revealed that DNA double-strand breaks (DSBs) elicit RNA-DNA hybrid formation. The RNA moiety of the R-loop displays an extraordinary potential to regulate multiple steps of homologous recombination (HR) by substituting for the double-stranded DNA (dsDNA) substrate on which DSB repair processes naturally occur. Here, we use the Damage At RNA Transcription assay to reveal colocalization of FANCA with R-loops in a highly transcribed genomic locus upon DNA damage. We further demonstrate FANCA participates in forming R-loops in the initial DSBs repair stage and assists in removing R-loops later. Besides, we find that highly purified human FANCA anneals synthetic single-stranded RNA (ssRNA) and ssDNA species to R-loops and binds R-loop substrates with high affinity, preferring guanine-rich sequences in vitro. Importantly, we illustrate that FANCA promotes HR efficiency via catalyzation of DNA: RNA hybrids at DSBs sites. Finally, a series of RNA and R-loop substrates are found to stimulate ID2 monoubiquitination, with activity corresponding to remove transient R- loops in later HR stage. In summary, our results support a mechanism whereby FANCA promotes the formation of temporary R-loops by annealing ssRNA and ssDNA species at DSBs in transcribed regions, thereby stimulating HR. Citation Format: Fang Li. FANCA promotes transcription-coupled homologous recombination by catalyzing R-loops formation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6214.

Developmentally Programmed Switches in DNA Replication: Gene Amplification and Genome-Wide Endoreplication in Tetrahymena.

Locus-specific gene amplification and genome-wide endoreplication generate the elevated copy number of ribosomal DNA (rDNA, 9000 C) and non-rDNA (90 C) chromosomes in the developing macronucleus of Tetrahymena thermophila. Subsequently, all macronuclear chromosomes replicate once per cell cycle during vegetative growth. Here, we describe an unanticipated, programmed switch in the regulation of replication initiation in the rDNA minichromosome. Early in development, the 21 kb rDNA minichromosome is preferentially amplified from 2 C to ~800 C from well-defined origins, concurrent with genome-wide endoreplication (2 C to 8-16 C) in starved mating Tetrahymena (endoreplication (ER) Phase 1). Upon refeeding, rDNA and non-rDNA chromosomes achieve their final copy number through resumption of just the endoreplication program (ER Phase 2). Unconventional rDNA replication intermediates are generated primarily during ER phase 2, consistent with delocalized replication initiation and possible formation of persistent RNA-DNA hybrids. Origin usage and replication fork elongation are affected in non-rDNA chromosomes as well. Despite the developmentally programmed 10-fold reduction in the ubiquitous eukaryotic initiator, the Origin Recognition Complex (ORC), active initiation sites are more closely spaced in ER phases 1 and 2 compared to vegetative growing cells. We propose that initiation site selection is relaxed in endoreplicating macronuclear chromosomes and may be less dependent on ORC.

Preprint Highlight: A role for phase separation to prevent R-loops during transcription.

The RNA-binding protein FUS (FUSed in sarcoma) regulates transcription by binding to polymerases and undergoing phase separation to enrich transcription machinery and RNA processing factors. Using an in vitro transcription assay and a DNA-dependent RNA polymerase from T7 phage (T7 Pol) that does not bind to FUS, this study uncovered a polymerase-binding independent role of FUS in increasing transcription productivity. The ability of FUS to phase separate with RNA prevented RNA-DNA hybrid (R-loop) formation during transcription in vitro and in cells. This work suggests a potential link between the toxicity of FUS mutants in amyotrophic lateral sclerosis or Ewing sarcoma to elevated R-loops and the resulting disruption in gene expression and genome stability.

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/CREB-binding protein (CBP) co-activator is associated with two-thirds of PcG regions and required for PcG occupancy at many of these in Drosophila and mouse cells. CBP stabilizes RNA polymerase II (Pol II) at PcG-bound repressive 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.

Chl1 coordinates with H3K9 methyltransferase Clr4 to reduce the accumulation of RNA-DNA hybrids and maintain genome stability

SummaryA genome-wide analysis in Schizosaccharomyces pombe indicated that double-deletion mutants of Chl1 and histone H3K9 methyltransferase complex factors are synthetically sick. Here, we show that loss of Chl1 increases the accumulation of RNA-DNA hybrids at pericentromeric dg and dh repeats in the absence of the H3K9 methyltransferase Clr4, which leads to genome instability, including more severe defects in chromosome segregation and increased chromatin accessibility. Localization of Chl1 at pericentromeric regions depends on a subunit of replication protein A (RPA), Ssb1. In wild-type (WT) cells, transcriptionally repressed heterochromatin prevents the formation of RNA-DNA hybrids. When Clr4 is deleted, dg and dh repeats are highly transcribed. Then Ssb1 associates with the displaced single-stranded DNA (ssDNA) and recruits Chl1 to resolve the RNA-DNA hybrids. Together, our data suggest that Chl1 coordinates with Clr4 to eliminate RNA-DNA hybrids, which contributes to the maintenance of genome integrity.

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.

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 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.

RNA-DNA strand exchange by the Drosophila Polycomb complex PRC2

Polycomb Group (PcG) proteins form memory of transient transcriptional repression that is necessary for development. In Drosophila, DNA elements termed Polycomb Response Elements (PREs) recruit PcG proteins. How PcG activities are targeted to PREs to maintain repressed states only in appropriate developmental contexts has been difficult to elucidate. PcG complexes modify chromatin, but also interact with both RNA and DNA, and RNA is implicated in PcG targeting and function. Here we show that R-loops form at many PREs in Drosophila embryos, and correlate with repressive states. In vitro, both PRC1 and PRC2 can recognize R-loops and open DNA bubbles. Unexpectedly, we find that PRC2 drives formation of RNA-DNA hybrids, the key component of R-loops, from RNA and dsDNA. Our results identify R-loop formation as a feature of Drosophila PREs that can be recognized by PcG complexes, and RNA-DNA strand exchange as a PRC2 activity that could contribute to R-loop formation.

Splicing events in the control of genome integrity: role of SLU7 and truncated SRSF3 proteins.

Genome instability is related to disease development and carcinogenesis. DNA lesions are caused by genotoxic compounds but also by the dysregulation of fundamental processes like transcription, DNA replication and mitosis. Recent evidence indicates that impaired expression of RNA-binding proteins results in mitotic aberrations and the formation of transcription-associated RNA–DNA hybrids (R-loops), events strongly associated with DNA injury. We identify the splicing regulator SLU7 as a key mediator of genome stability. SLU7 knockdown results in R-loops formation, DNA damage, cell-cycle arrest and severe mitotic derangements with loss of sister chromatid cohesion (SCC). We define a molecular pathway through which SLU7 keeps in check the generation of truncated forms of the splicing factor SRSF3 (SRp20) (SRSF3-TR). Behaving as dominant negative, or by gain-of-function, SRSF3-TR impair the correct splicing and expression of the splicing regulator SRSF1 (ASF/SF2) and the crucial SCC protein sororin. This unique function of SLU7 was found in cancer cells of different tissue origin and also in the normal mouse liver, demonstrating a conserved and fundamental role of SLU7 in the preservation of genome integrity. Therefore, the dowregulation of SLU7 and the alterations of this pathway that we observe in the cirrhotic liver could be involved in the process of hepatocarcinogenesis.

Heterochromatin-Encoded Satellite RNAs Induce Breast Cancer

Heterochromatic repetitive satellite RNAs are extensively transcribed in a variety of human cancers, including BRCA1 mutant breast cancer. Aberrant expression of satellite RNAs in cultured cells induces the DNA damage response, activates cell cycle checkpoints, and causes defects in chromosome segregation. However, the mechanism by which satellite RNA expression leads to genomic instability is not well understood. Here we provide evidence that increased levels of satellite RNAs in mammary glands induce tumor formation in mice. Using mass spectrometry, we further show that genomic instability induced by satellite RNAs occurs through interactions with BRCA1-associated protein networks required for the stabilization of DNA replication forks. Additionally, de-stabilized replication forks likely promote the formation of RNA-DNA hybrids in cells expressing satellite RNAs. These studies lay the foundation for developing novel therapeutic strategies that block the effects of non-coding satellite RNAs in cancer cells.

Genome-wide relationship between R-loop formation and antisense transcription in Escherichia coli.

Transcription termination by Rho is essential for viability in various bacteria, including some major pathogens. Since Rho acts by targeting nascent RNAs that are not simultaneously translated, it also regulates antisense transcription. Here we show that RNase H-deficient mutants of Escherichia coli exhibit heightened sensitivity to the Rho inhibitor bicyclomycin, and that Rho deficiency provokes increased formation of RNA–DNA hybrids (R-loops) which is ameliorated by expression of the phage T4-derived R-loop helicase UvsW. We also provide evidence that in Rho-deficient cells, R-loop formation blocks subsequent rounds of antisense transcription at more than 500 chromosomal loci. Hence these antisense transcripts, which can extend beyond 10 kb in their length, are only detected when Rho function is absent or compromised and the UvsW helicase is concurrently expressed. Thus the potential for antisense transcription in bacteria is much greater than hitherto recognized; and the cells are able to retain viability even when nearly one-quarter of their total non-rRNA abundance is accounted for by antisense transcripts, provided that R-loop formation from them is curtailed.

RNA-DNA hybrids promote the expansion of Friedreich's ataxia(GAA)n repeats via break-induced replication.

Expansion of simple DNA repeats is responsible for numerous hereditary diseases in humans. The role of DNA replication, repair and transcription in the expansion process has been well documented. Here we analyzed, in a yeast experimental system, the role of RNA–DNA hybrids in genetic instability of long (GAA)n repeats, which cause Friedreich’s ataxia. Knocking out both yeast RNase H enzymes, which counteract the formation of RNA–DNA hybrids, increased (GAA)n repeat expansion and contraction rates when the repetitive sequence was transcribed. Unexpectedly, we observed a similar increase in repeat instability in RNase H-deficient cells when we either changed the direction of transcription-replication collisions, or flipped the repeat sequence such that the (UUC)n run occurred in the transcript. The increase in repeat expansions in RNase H-deficient strains was dependent on Rad52 and Pol32 proteins, suggesting that break-induced replication (BIR) is responsible for this effect. We conclude that expansions of (GAA)n repeats are induced by the formation of RNA–DNA hybrids that trigger BIR. Since this stimulation is independent of which strand of the repeat (homopurine or homopyrimidine) is in the RNA transcript, we hypothesize that triplex H-DNA structures stabilized by an RNA–DNA hybrid (H-loops), rather than conventional R-loops, could be responsible.

TRF1 participates in chromosome end protection by averting TRF2-dependent telomeric R loops.

The shelterin protein TRF2 assembles protective T-loops at chromosome ends by stimulating intramolecular invasion of the telomeric G-rich ssDNA overhang into the duplex telomeric array. The other shelterin factor TRF1 is thought to mainly facilitate telomeric dsDNA replication without directly participating in end protection. Here we show that in vitro human TRF2 stimulates invasion of G-rich TERRA-like RNA into telomeric dsDNA leading to formation of telomeric RNA:DNA hybrids (telR-loops). The N-terminal basic domain of TRF2 binds to TERRA-like RNA and enables TRF2 to promote efficient RNA invasion. TRF1, through its N-terminal acidic domain, counteracts TRF2-mediated RNA but not ssDNA invasion. In vivo, when TRF1 is depleted or replaced with a variant lacking the acidic domain, TRF2 induces formation of telR-loops, which in turn cause telomere loss. Hence, uncontrolled TRF2 threatens telomere integrity and TRF1 directly supports end protection by suppressing harmful telR-loops.

Endogenous LINE-1 (Long Interspersed Nuclear Element-1) Reverse Transcriptase Activity in Platelets Controls Translational Events Through RNA-DNA Hybrids.

One source of endogenous reverse transcriptase (eRT) activity in nucleated cells is the LINE-1/L1 (long interspersed nuclear element-1), a non-LTR retrotransposon that is implicated in the regulation of gene expression. Nevertheless, the presence and function of eRT activity and LINE-1 in human platelets, an anucleate cell, has not previously been determined. We demonstrate that human and murine platelets possess robust eRT activity and identify the source as being LINE-1 ribonucleoprotein particles. Inhibition of eRT in vitro in isolated platelets from healthy individuals or in people with HIV treated with RT inhibitors enhanced global protein synthesis and platelet activation. If HIV patients were treated with reverse transcriptase inhibitor, we found that platelets from these patients had increased basal activation. We next discovered that eRT activity in platelets controlled the generation of RNA-DNA hybrids, which serve as translational repressors. Inhibition of platelet eRT lifted this RNA-DNA hybrid-induced translational block and was sufficient to increase protein expression of target RNAs identified by RNA-DNA hybrid immunoprecipitation. Thus, we provide the first evidence that platelets possess L1-encoded eRT activity. We also demonstrate that platelet eRT activity regulates platelet hyperreactivity and thrombosis and controls RNA-DNA hybrid formation and identify that RNA-DNA hybrids function as a novel translational control mechanism in human platelets.

Transcription-Replication Conflicts: Orientation Matters

Interference between DNA replication and transcription represents a major source of genomic instability. In this issue of Cell, Lang etal. and Hamperl etal. show that head-on collisions, but not codirectional collisions, impede fork progression in bacteria and in human cells by promoting the formation of RNA-DNA hybrids known as R-loops.

Strong transcription blockage mediated by R-loop formation within a G-rich homopurine-homopyrimidine sequence localized in the vicinity of the promoter.

Guanine-rich (G-rich) homopurine–homopyrimidine nucleotide sequences can block transcription with an efficiency that depends upon their orientation, composition and length, as well as the presence of negative supercoiling or breaks in the non-template DNA strand. We report that a G-rich sequence in the non-template strand reduces the yield of T7 RNA polymerase transcription by more than an order of magnitude when positioned close (9 bp) to the promoter, in comparison to that for a distal (∼250 bp) location of the same sequence. This transcription blockage is much less pronounced for a C-rich sequence, and is not significant for an A-rich sequence. Remarkably, the blockage is not pronounced if transcription is performed in the presence of RNase H, which specifically digests the RNA strands within RNA–DNA hybrids. The blockage also becomes less pronounced upon reduced RNA polymerase concentration. Based upon these observations and those from control experiments, we conclude that the blockage is primarily due to the formation of stable RNA–DNA hybrids (R-loops), which inhibit successive rounds of transcription. Our results could be relevant to transcription dynamics in vivo (e.g. transcription ‘bursting’) and may also have practical implications for the design of expression vectors.

LncRNAs promote gene expression, too

GAL lncRNAs in yeast induce gene transcription by enabling gene looping through the formation of RNA-DNA hybrids.

The spliceosome-associated protein Nrl1 suppresses homologous recombination-dependent R-loop formation in fission yeast.

The formation of RNA–DNA hybrids, referred to as R-loops, can promote genome instability and cancer development. Yet the mechanisms by which R-loops compromise genome instability are poorly understood. Here, we establish roles for the evolutionarily conserved Nrl1 protein in pre-mRNA splicing regulation, R-loop suppression and in maintaining genome stability. nrl1Δ mutants exhibit endogenous DNA damage, are sensitive to exogenous DNA damage, and have defects in homologous recombination (HR) repair. Concomitantly, nrl1Δ cells display significant changes in gene expression, similar to those induced by DNA damage in wild-type cells. Further, we find that nrl1Δ cells accumulate high levels of R-loops, which co-localize with HR repair factors and require Rad51 and Rad52 for their formation. Together, our findings support a model in which R-loop accumulation and subsequent DNA damage sequesters HR factors, thereby compromising HR repair at endogenously or exogenously induced DNA damage sites, leading to genome instability.