RESUMEN
The specific nature of CRISPR-Cas12a makes it a desirable RNA-guided endonuclease for biotechnology and therapeutic applications. To understand how R-loop formation within the compact Cas12a enables target recognition and nuclease activation, we used cryo-electron microscopy to capture wild-type Acidaminococcus sp. Cas12a R-loop intermediates and DNA delivery into the RuvC active site. Stages of Cas12a R-loop formation-starting from a 5-bp seed-are marked by distinct REC domain arrangements. Dramatic domain flexibility limits contacts until nearly complete R-loop formation, when the non-target strand is pulled across the RuvC nuclease and coordinated domain docking promotes efficient cleavage. Next, substantial domain movements enable target strand repositioning into the RuvC active site. Between cleavage events, the RuvC lid conformationally resets to occlude the active site, requiring re-activation. These snapshots build a structural model depicting Cas12a DNA targeting that rationalizes observed specificity and highlights mechanistic comparisons to other class 2 effectors.
Asunto(s)
Acidaminococcus , Proteínas Bacterianas , Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , Dominio Catalítico , Microscopía por Crioelectrón , Proteínas Asociadas a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/genética , Acidaminococcus/enzimología , Acidaminococcus/genética , Acidaminococcus/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Estructuras R-Loop/genética , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/química , ARN Guía de Sistemas CRISPR-Cas/metabolismo , ARN Guía de Sistemas CRISPR-Cas/genética , Modelos Moleculares , Dominios Proteicos , Relación Estructura-Actividad , Unión ProteicaRESUMEN
Mutations in the Cockayne Syndrome group B (CSB) gene cause cancer in mice, but premature aging and severe neurodevelopmental defects in humans. CSB, a member of the SWI/SNF family of chromatin remodelers, plays diverse roles in regulating gene expression and transcription-coupled nucleotide excision repair (TC-NER); however, these functions do not explain the distinct phenotypic differences observed between CSB-deficient mice and humans. During investigating Cockayne Syndrome-associated genome instability, we uncover an intrinsic mechanism that involves elongating RNA polymerase II (RNAPII) undergoing transient pauses at internal T-runs where CSB is required to propel RNAPII forward. Consequently, CSB deficiency retards RNAPII elongation in these regions, and when coupled with G-rich sequences upstream, exacerbates genome instability by promoting R-loop formation. These R-loop prone motifs are notably abundant in relatively long genes related to neuronal functions in the human genome, but less prevalent in the mouse genome. These findings provide mechanistic insights into differential impacts of CSB deficiency on mice versus humans and suggest that the manifestation of the Cockayne Syndrome phenotype in humans results from the progressive evolution of mammalian genomes.
Asunto(s)
Síndrome de Cockayne , ADN Helicasas , Enzimas Reparadoras del ADN , Inestabilidad Genómica , Proteínas de Unión a Poli-ADP-Ribosa , Estructuras R-Loop , ARN Polimerasa II , Síndrome de Cockayne/genética , Síndrome de Cockayne/patología , Síndrome de Cockayne/metabolismo , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Animales , Humanos , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Ratones , ADN Helicasas/metabolismo , ADN Helicasas/genética , Estructuras R-Loop/genética , Reparación del ADN , Elongación de la Transcripción Genética , Ratones NoqueadosRESUMEN
Three-stranded DNA-RNA structures known as R-loops that form during papillomavirus transcription can cause transcription-replication conflicts and lead to DNA damage. We found that R-loops accumulated at the viral early promoter in human papillomavirus (HPV) episomal cells but were greatly reduced in cells with integrated HPV genomes. RNA-DNA helicases unwind R-loops and allow for transcription and replication to proceed. Depletion of the RNA-DNA helicase senataxin (SETX) using siRNAs increased the presence of R-loops at the viral early promoter in HPV-31 (CIN612) and HPV-16 (W12) episomal HPV cell lines. Depletion of SETX reduced viral transcripts in episomal HPV cell lines. The viral E2 protein, which binds with high affinity to specific palindromes near the promoter and origin, complexes with SETX, and both SETX and E2 are present at the viral p97 promoter in CIN612 and W12 cells. SETX overexpression increased E2 transcription activity on the p97 promoter. SETX depletion also significantly increased integration of viral genomes in CIN612 cells. Our results demonstrate that SETX resolves viral R-loops to proceed with HPV transcription and prevent genome integration.IMPORTANCEPapillomaviruses contain small circular genomes of approximately 8 kilobase pairs and undergo unidirectional transcription from the sense strand of the viral genome. Co-transcriptional R-loops were recently reported to be present at high levels in cells that maintain episomal HPV and were also detected at the early viral promoter. R-loops can inhibit transcription and DNA replication. The process that removes R-loops from the PV genome and the requisite enzymes are unknown. We propose a model in which the host RNA-DNA helicase senataxin assembles on the HPV genome to resolve R-loops in order to maintain the episomal status of the viral genome.
Asunto(s)
ADN Helicasas , Enzimas Multifuncionales , Regiones Promotoras Genéticas , Estructuras R-Loop , ARN Helicasas , Humanos , ARN Helicasas/genética , ARN Helicasas/metabolismo , Enzimas Multifuncionales/genética , Enzimas Multifuncionales/metabolismo , ADN Helicasas/metabolismo , ADN Helicasas/genética , Estructuras R-Loop/genética , Plásmidos/genética , Replicación Viral , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Papillomaviridae/genética , Genoma Viral , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Transcripción Genética , Línea Celular , ADN Viral/genéticaRESUMEN
R-loops are three-stranded nucleic acid structures that are abundant and widespread across the genome and that have important physiological roles in many nuclear processes. Their accumulation is observed in cancers and neurodegenerative disorders. Recent studies have implicated a function for R-loops and G-quadruplex (G4) structures, which can form on the displaced single strand of R-loops, in three-dimensional genome organization in both physiological and pathological contexts. Here we discuss the interconnected functions of DNA:RNA hybrids and G4s within R-loops, their impact on DNA repair and gene regulatory networks, and their emerging roles in genome organization during development and disease.
Asunto(s)
Reparación del ADN , G-Cuádruplex , Estructuras R-Loop , Transcripción Genética , Humanos , Estructuras R-Loop/genética , Animales , ADN/metabolismo , ADN/genética , ADN/química , Genoma/genética , Redes Reguladoras de Genes , ARN/metabolismo , ARN/genética , ARN/química , Neoplasias/genética , Neoplasias/patología , Neoplasias/metabolismoRESUMEN
CircR-loop, a recently unearthed regulatory mechanism situated at the crossroads of circular RNA and DNA interactions, constitute a subset of R-loop. This circR-loop have emerged as a crucial player in pivotal regulatory functions within both animal and plant systems. The journey into the realm of circR-loop commenced with their discovery within the human mitochondrial genome, where they serve as critical directors of mitochondrial DNA replication. In the plant kingdom, circR-loop wield influence over processes such as alternative splicing and centromere organization, impacting the intricacies of floral development and genome stability, respectively. Their significance extends to the animal domain, where circR-loop has captured attention for their roles in cancer-related phenomena, exerting control over transcription, chromatin architecture, and orchestrating responses to DNA damage. Moreover, their involvement in nuclear export anomalies further underscores their prominence in cellular regulation. This article summarizes the important regulatory mechanisms and physiological roles of circR-loop in plants and animals, and offers a comprehensive exploration of the methodologies employed for the identification, characterization, and functional analysis of circR-loop, underscoring the pressing need for innovative approaches that can effectively distinguish them from their linear RNA counterparts while elucidating their precise functions. Lastly, the article sheds light on the challenges and opportunities that lie ahead in the field of circR-loop research, emphasizing the vital importance of continued investigations to uncover their regulatory roles and potential applications in the realm of biology. In summary, circR-loop represents a captivating and novel regulatory mechanism with broad-reaching implications spanning the realms of genetics, epigenetics, and disease biology. Their exploration opens new avenues for comprehending gene regulation and holds significant promise for future therapeutic interventions.
Asunto(s)
Inestabilidad Genómica , ARN Circular , Inestabilidad Genómica/genética , Humanos , Animales , ARN Circular/genética , ARN Circular/metabolismo , ADN/metabolismo , ADN/genética , Estructuras R-Loop/genética , ARN/metabolismo , ARN/genética , Replicación del ADN/genéticaRESUMEN
MCM8 has emerged as a core gene in reproductive aging and is crucial for meiotic homologous recombination repair. It also safeguards genome stability by coordinating the replication stress response during mitosis, but its function in mitotic germ cells remains elusive. Here we found that disabling MCM8 in mice resulted in proliferation defects of primordial germ cells (PGCs) and ultimately impaired fertility. We further demonstrated that MCM8 interacted with two known helicases DDX5 and DHX9, and loss of MCM8 led to R-loop accumulation by reducing the retention of these helicases at R-loops, thus inducing genome instability. Cells expressing premature ovarian insufficiency-causative mutants of MCM8 with decreased interaction with DDX5 displayed increased R-loop levels. These results show MCM8 interacts with R-loop-resolving factors to prevent R-loop-induced DNA damage, which may contribute to the maintenance of genome integrity of PGCs and reproductive reserve establishment. Our findings thus reveal an essential role for MCM8 in PGC development and improve our understanding of reproductive aging caused by genome instability in mitotic germ cells.
Asunto(s)
ARN Helicasas DEAD-box , Inestabilidad Genómica , Proteínas de Mantenimiento de Minicromosoma , Estructuras R-Loop , Animales , Femenino , Humanos , Masculino , Ratones , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Daño del ADN , Células Germinativas/metabolismo , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas de Mantenimiento de Minicromosoma/genética , Estructuras R-Loop/genéticaRESUMEN
Type I CRISPR-Cas systems employ multi-subunit effector Cascade and helicase-nuclease Cas3 to target and degrade foreign nucleic acids, representing the most abundant RNA-guided adaptive immune systems in prokaryotes. Their ability to cause long fragment deletions have led to increasing interests in eukaryotic genome editing. While the Cascade structures of all other six type I systems have been determined, the structure of the most evolutionarily conserved type I-B Cascade is still missing. Here, we present two cryo-EM structures of the Synechocystis sp. PCC 6714 (Syn) type I-B Cascade, revealing the molecular mechanisms that underlie RNA-directed Cascade assembly, target DNA recognition, and local conformational changes of the effector complex upon R-loop formation. Remarkably, a loop of Cas5 directly intercalated into the major groove of the PAM and facilitated PAM recognition. We further characterized the genome editing profiles of this I-B Cascade-Cas3 in human CD3+ T cells using mRNA-mediated delivery, which led to unidirectional 4.5 kb deletion in TRAC locus and achieved an editing efficiency up to 41.2%. Our study provides the structural basis for understanding target DNA recognition by type I-B Cascade and lays foundation for harnessing this system for long range genome editing in human T cells.
Asunto(s)
Sistemas CRISPR-Cas , Microscopía por Crioelectrón , Edición Génica , Synechocystis , Edición Génica/métodos , Humanos , Synechocystis/genética , Proteínas Asociadas a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Linfocitos T/metabolismo , Estructuras R-Loop/genéticaRESUMEN
Senataxin is an evolutionarily conserved RNA-DNA helicase involved in DNA repair and transcription termination that is associated with human neurodegenerative disorders. Here, we investigated whether Senataxin loss affects protein homeostasis based on previous work showing R-loop-driven accumulation of DNA damage and protein aggregates in human cells. We find that Senataxin loss results in the accumulation of insoluble proteins, including many factors known to be prone to aggregation in neurodegenerative disorders. These aggregates are located primarily in the nucleolus and are promoted by upregulation of non-coding RNAs expressed from the intergenic spacer region of ribosomal DNA. We also map sites of R-loop accumulation in human cells lacking Senataxin and find higher RNA-DNA hybrids within the ribosomal DNA, peri-centromeric regions, and other intergenic sites but not at annotated protein-coding genes. These findings indicate that Senataxin loss affects the solubility of the proteome through the regulation of transcription-dependent lesions in the nucleus and the nucleolus.
Asunto(s)
ADN Helicasas , Enzimas Multifuncionales , ARN Helicasas , ARN no Traducido , Humanos , Nucléolo Celular/metabolismo , Nucléolo Celular/genética , Daño del ADN , ADN Helicasas/metabolismo , ADN Helicasas/genética , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Enzimas Multifuncionales/metabolismo , Enzimas Multifuncionales/genética , Agregado de Proteínas , Proteostasis , Estructuras R-Loop/genética , ARN Helicasas/metabolismo , ARN Helicasas/genética , ARN no Traducido/genética , ARN no Traducido/metabolismoRESUMEN
As aberrant accumulation of RNA-DNA hybrids (R-loops) causes DNA damage and genome instability, cells express regulators of R-loop structures. Here we report that RNA-dependent RNA polymerase (RdRP) activity of human telomerase reverse transcriptase (hTERT) regulates R-loop formation. We found that the phosphorylated form of hTERT (p-hTERT) exhibits RdRP activity in nuclear speckles both in telomerase-positive cells and telomerase-negative cells with alternative lengthening of telomeres (ALT) activity. The p-hTERT did not associate with telomerase RNA component in nuclear speckles but, instead, with TERRA RNAs to resolve R-loops. Targeting of the TERT gene in ALT cells ablated RdRP activity and impaired tumour growth. Using a genome-scale CRISPR loss-of-function screen, we identified Fanconi anaemia/BRCA genes as synthetic lethal partners of hTERT RdRP. Inactivation of RdRP and Fanconi anaemia/BRCA genes caused accumulation of R-loop structures and DNA damage. These findings indicate that RdRP activity of p-hTERT guards against genome instability by removing R-loop structures.
Asunto(s)
Daño del ADN , Inestabilidad Genómica , Estructuras R-Loop , Telomerasa , Homeostasis del Telómero , Telomerasa/genética , Telomerasa/metabolismo , Humanos , Fosforilación , Inestabilidad Genómica/genética , Estructuras R-Loop/genética , ARN/metabolismo , ARN/genética , Animales , Células HEK293 , Telómero/metabolismo , Telómero/genética , Línea Celular TumoralRESUMEN
Certain DNA sequences can adopt a non-B form in the genome that interfere with DNA-templated processes, including transcription. Among the sequences that are intrinsically difficult to transcribe are those that tend to form R-loops, three-stranded nucleic acid structures formed by a DNA-RNA hybrid and the displaced ssDNA. Here we compared the transcription of an endogenous gene with and without an R-loop-forming sequence inserted. We show that, in agreement with previous in vivo and in vitro analyses, transcription elongation is delayed by R-loops in yeast. Importantly, we demonstrate that the Rat1 transcription terminator factor facilitates transcription throughout such structures by inducing premature termination of arrested RNAPIIs. We propose that RNase H degrades the RNA moiety of the hybrid, providing an entry site for Rat1. Thus, we have uncovered an unanticipated function of Rat1 as a transcription restoring factor opening up the possibility that it may also promote transcription through other genomic DNA structures intrinsically difficult to transcribe. If R-loop-mediated transcriptional stress is not relieved by Rat1, it will cause genomic instability, probably through the increase of transcription-replication conflicts, a deleterious situation that could lead to cancer.
Asunto(s)
Exorribonucleasas , Estructuras R-Loop , Ribonucleasa H , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Terminación de la Transcripción Genética , Estructuras R-Loop/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ribonucleasa H/metabolismo , Ribonucleasa H/genética , Saccharomyces cerevisiae/genética , ARN Polimerasa II/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
R-loops that accumulate at transcription sites pose a persistent threat to genome integrity. PSIP1 is a chromatin protein associated with transcriptional elongation complex, possesses histone chaperone activity, and is implicated in recruiting RNA processing and DNA repair factors to transcription sites. Here, we show that PSIP1 interacts with R-loops and other proteins involved in R-loop homeostasis, including PARP1. Genome-wide mapping of PSIP1, R-loops and γ-H2AX in PSIP1-depleted human and mouse cell lines revealed an accumulation of R-loops and DNA damage at gene promoters in the absence of PSIP1. R-loop accumulation causes local transcriptional arrest and transcription-replication conflict, leading to DNA damage. PSIP1 depletion increases 53BP1 foci and reduces RAD51 foci, suggesting altered DNA repair choice. Furthermore, PSIP1 depletion increases the sensitivity of cancer cells to PARP1 inhibitors and DNA-damaging agents that induce R-loop-induced DNA damage. These findings provide insights into the mechanism through which PSIP1 maintains genome integrity at the site of transcription.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular , Estructuras R-Loop , Humanos , Animales , Ratones , Estructuras R-Loop/genética , Línea Celular , Daño del ADN , Factores de Transcripción/genética , Proteínas Adaptadoras Transductoras de SeñalesRESUMEN
Genome topology is tied to R-loop formation and genome stability. However, the regulatory mechanism remains to be elucidated. By establishing a system to sense the connections between R-loops and genome topology states, we show that inhibiting DNA topoisomerase 1 (TOP1i) triggers the global increase of R-loops (called topoR-loops) and DNA damages, which are exacerbated in the DNA damage repair-compromised mutant atm. A suppressor screen identifies a mutation in POL2A, the catalytic subunit of DNA polymerase ε, rescuing the TOP1i-induced topoR-loop accumulation and genome instability in atm. Importantly we find that a highly conserved junction domain between the exonuclease and polymerase domains in POL2A is required for modulating topoR-loops near DNA replication origins and facilitating faithful DNA replication. Our results suggest that DNA replication acts in concert with genome topological states to fine-tune R-loops and thereby maintain genome integrity, revealing a likely conserved regulatory mechanism of TOP1i resistance in chemotherapy for ATM-deficient cancers.
Asunto(s)
Arabidopsis , Humanos , Arabidopsis/genética , Estructuras R-Loop/genética , ADN Polimerasa II/genética , Replicación del ADN/genética , Mutación , Daño del ADN , Inestabilidad Genómica/genéticaRESUMEN
R-loops, which consist of a DNA-RNA hybrid and a displaced DNA strand, are known to threaten genome integrity. To counteract this, different mechanisms suppress R-loop accumulation by either preventing the hybridization of RNA with the DNA template (RNA biogenesis factors), unwinding the hybrid (DNA-RNA helicases), or degrading the RNA moiety of the R-loop (type H ribonucleases [RNases H]). Thus far, RNases H are the only nucleases known to cleave DNA-RNA hybrids. Now, we show that the RNase DICER also resolves R-loops. Biochemical analysis reveals that DICER acts by specifically cleaving the RNA within R-loops. Importantly, a DICER RNase mutant impaired in R-loop processing causes a strong accumulation of R-loops in cells. Our results thus not only reveal a function of DICER as an R-loop resolvase independent of DROSHA but also provide evidence for the role of multi-functional RNA processing factors in the maintenance of genome integrity in higher eukaryotes.
Asunto(s)
Estructuras R-Loop , Ribonucleasas , Humanos , Estructuras R-Loop/genética , Ribonucleasas/genética , ARN/genética , ADN , Replicación del ADN , ADN Helicasas/genética , Ribonucleasa H/genética , Ribonucleasa H/metabolismo , Inestabilidad GenómicaRESUMEN
The protein FUS (FUSed in sarcoma) is a metazoan RNA-binding protein that influences RNA production by all three nuclear polymerases. FUS also binds nascent transcripts, RNA processing factors, RNA polymerases, and transcription machinery. Here, we explored the role of FUS binding interactions for activity during transcription. In vitro run-off transcription assays revealed FUS-enhanced RNA produced by a non-eukaryote polymerase. The activity also reduced the formation of R-loops between RNA products and their DNA template. Analysis by domain mutation and deletion indicated RNA-binding was required for activity. We interpret that FUS binds and sequesters nascent transcripts to prevent R-loops from forming with nearby DNA. DRIP-seq analysis showed that a knockdown of FUS increased R-loop enrichment near expressed genes. Prevention of R-loops by FUS binding to nascent transcripts has the potential to affect transcription by any RNA polymerase, highlighting the broad impact FUS can have on RNA metabolism in cells and disease.
Asunto(s)
ADN , Estructuras R-Loop , Proteína FUS de Unión a ARN , ARN , ADN/metabolismo , Estructuras R-Loop/genética , ARN/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Unión Proteica , Humanos , ARN Polimerasas Dirigidas por ADN/metabolismo , Células HEK293RESUMEN
The roles of R-loops and RNA modifications in homologous recombination (HR) and other DNA double-stranded break (DSB) repair pathways remain poorly understood. Here, we find that DNA damage-induced RNA methyl-5-cytosine (m5C) modification in R-loops plays a crucial role to regulate PARP1-mediated poly ADP-ribosylation (PARylation) and the choice of DSB repair pathways at sites of R-loops. Through bisulfite sequencing, we discover that the methyltransferase TRDMT1 preferentially generates m5C after DNA damage in R-loops across the genome. In the absence of m5C, R-loops activate PARP1-mediated PARylation both in vitro and in cells. Concurrently, m5C promotes transcription-coupled HR (TC-HR) while suppressing PARP1-dependent alternative non-homologous end joining (Alt-NHEJ), favoring TC-HR over Alt-NHEJ in transcribed regions as the preferred repair pathway. Importantly, simultaneous disruption of both TC-HR and Alt-NHEJ with TRDMT1 and PARP or Polymerase θ inhibitors prevents alternative DSB repair and exhibits synergistic cytotoxic effects on cancer cells, suggesting an effective strategy to exploit genomic instability in cancer therapy.
Asunto(s)
Citosina , Estructuras R-Loop , Estructuras R-Loop/genética , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , ARN/genética , Reparación del ADNRESUMEN
While the toxicity of PARP inhibitors to cells with defects in homologous recombination (HR) is well established, other synthetic lethal interactions with PARP1/PARP2 disruption are poorly defined. To inform on these mechanisms we conducted a genome-wide screen for genes that are synthetic lethal with PARP1/2 gene disruption and identified C16orf72/HAPSTR1/TAPR1 as a novel modulator of replication-associated R-loops. C16orf72 is critical to facilitate replication fork restart, suppress DNA damage and maintain genome stability in response to replication stress. Importantly, C16orf72 and PARP1/2 function in parallel pathways to suppress DNA:RNA hybrids that accumulate at stalled replication forks. Mechanistically, this is achieved through an interaction of C16orf72 with BRCA1 and the RNA/DNA helicase Senataxin to facilitate their recruitment to RNA:DNA hybrids and confer resistance to PARP inhibitors. Together, this identifies a C16orf72/Senataxin/BRCA1-dependent pathway to suppress replication-associated R-loop accumulation, maintain genome stability and confer resistance to PARP inhibitors.
Asunto(s)
Proteína BRCA1 , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Estructuras R-Loop , Daño del ADN , ADN Helicasas/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Estructuras R-Loop/genética , ARN , Proteína BRCA1/genética , Péptidos y Proteínas de Señalización Intracelular/genéticaRESUMEN
The proper regulation of transcription is essential for maintaining genome integrity and executing other downstream cellular functions1,2. Here we identify a stable association between the genome-stability regulator sensor of single-stranded DNA (SOSS)3 and the transcription regulator Integrator-PP2A (INTAC)4-6. Through SSB1-mediated recognition of single-stranded DNA, SOSS-INTAC stimulates promoter-proximal termination of transcription and attenuates R-loops associated with paused RNA polymerase II to prevent R-loop-induced genome instability. SOSS-INTAC-dependent attenuation of R-loops is enhanced by the ability of SSB1 to form liquid-like condensates. Deletion of NABP2 (encoding SSB1) or introduction of cancer-associated mutations into its intrinsically disordered region leads to a pervasive accumulation of R-loops, highlighting a genome surveillance function of SOSS-INTAC that enables timely termination of transcription at promoters to constrain R-loop accumulation and ensure genome stability.
Asunto(s)
Inestabilidad Genómica , Regiones Promotoras Genéticas , Estructuras R-Loop , Terminación de la Transcripción Genética , Humanos , ADN de Cadena Simple/metabolismo , Inestabilidad Genómica/genética , Mutación , Estructuras R-Loop/genética , ARN Polimerasa II/metabolismo , Regiones Promotoras Genéticas/genética , Genoma Humano , Proteínas de Unión al ADN/metabolismoRESUMEN
The prototype enzymes of the ubiquitous type IA topoisomerases (topos) family are Escherichia coli topo I (topA) and topo III (topB). Topo I shows preference for relaxation of negative supercoiling and topo III for decatenation. However, as they could act as backups for each other or even share functions, strains lacking both enzymes must be used to reveal the roles of type IA enzymes in genome maintenance. Recently, marker frequency analysis (MFA) of genomic DNA from topA topB null mutants revealed a major RNase HI-sensitive DNA peak bordered by Ter/Tus barriers, sites of replication fork fusion and termination in the chromosome terminus region (Ter). Here, flow cytometry for R-loop-dependent replication (RLDR), MFA, R-loop detection with S9.6 antibodies, and microscopy were used to further characterize the mechanism and consequences of over-replication in Ter. It is shown that the Ter peak is not due to the presence of a strong origin for RLDR in Ter region; instead RLDR, which is partly inhibited by the backtracking-resistant rpoB*35 mutation, appears to contribute indirectly to Ter over-replication. The data suggest that RLDR from multiple sites on the chromosome increases the number of replication forks trapped at Ter/Tus barriers which leads to RecA-dependent DNA amplification in Ter and to a chromosome segregation defect. Overproducing topo IV, the main cellular decatenase, does not inhibit RLDR or Ter over-replication but corrects the chromosome segregation defect. Furthermore, our data suggest that the inhibition of RLDR by topo I does not require its C-terminal-mediated interaction with RNA polymerase. Overall, our data reveal a pathway of genomic instability triggered by R-loops and its regulation by various topos activities at different steps.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Humanos , Escherichia coli/metabolismo , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/metabolismo , Replicación del ADN/genética , Estructuras R-Loop/genética , Inestabilidad Genómica/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismoRESUMEN
R-loops are DNA-RNA hybrids that play multifunctional roles in gene regulation, including replication, transcription, transcription-replication collision, epigenetics, and preserving the integrity of the genome. The aberrant formation and accumulation of unscheduled R-loops can disrupt gene expression and damage DNA, thereby causing genome instability. Recent links between unscheduled R-loop accumulation and the abundance of proteins that modulate R-loop biogenesis have been associated with numerous human diseases, including various cancers. Although R-loops are not necessarily causative for all disease entities described to date, they can perpetuate and even exacerbate the initially disease-eliciting pathophysiology, making them structures of interest for molecular diagnostics. In this review, we discuss the (patho) physiological role of R-loops in health and disease, their surprising diagnostic potential, and state-of-the-art techniques for their detection.
Asunto(s)
Neoplasias , Estructuras R-Loop , Humanos , Estructuras R-Loop/genética , Neoplasias/diagnóstico , Neoplasias/genética , ADN/genética , Regulación de la Expresión Génica , ARN/genéticaRESUMEN
R-loops affect transcription and genome stability. Dysregulation of R-loops is related to human diseases. Genome-wide R-loop mapping typically uses the S9.6 antibody or inactive ribonuclease H, both requiring a large number of cells with varying results observed depending on the approach applied. Here, we present strand-specific kethoxal-assisted single-stranded DNA (ssDNA) sequencing (spKAS-seq) to map R-loops by taking advantage of the presence of a ssDNA in the triplex structure. We show that spKAS-seq detects R-loops and their dynamics at coding sequences, enhancers, and other intergenic regions with as few as 50,000 cells. A joint analysis of R-loops and chromatin-bound RNA binding proteins (RBPs) suggested that R-loops can be RBP binding hotspots on the chromatin.