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1.
J Vis Exp ; (174)2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34515688

RESUMO

R-loops constitute a prevalent class of transcription-driven non-B DNA structures that occur in all genomes depending on both DNA sequence and topological favorability. In recent years, R-loops have been implicated in a variety of adaptive and maladaptive roles and have been linked to genomic instability in the context of human disorders. As a consequence, the accurate mapping of these structures in genomes is of high interest to many investigators. DRIP-seq (DNA:RNA Immunoprecipitation followed by high throughput sequencing) is described here. It is a robust and reproducible technique that permits accurate and semi-quantitative mapping of R-loops. A recent iteration of the method is also described in which fragmentation is accomplished using sonication (sDRIP-seq), which allows strand-specific and high-resolution mapping of R-loops. sDRIP-seq thus addresses some of the common limitations of the DRIP-seq method in terms of resolution and strandedness, making it a method of choice for R-loop mapping.


Assuntos
Estruturas R-Loop , RNA , DNA/genética , Técnicas Genéticas , Instabilidade Genômica , Humanos , Imunoprecipitação , RNA/genética , Transcrição Genética
3.
Int J Mol Sci ; 22(16)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34445553

RESUMO

Since their discovery, R-loops have been associated with both physiological and pathological functions that are conserved across species. R-loops are a source of replication stress and genome instability, as seen in neurodegenerative disorders and cancer. In response, cells have evolved pathways to prevent R-loop accumulation as well as to resolve them. A growing body of evidence correlates R-loop accumulation with changes in the epigenetic landscape. However, the role of chromatin modification and remodeling in R-loops homeostasis remains unclear. This review covers various mechanisms precluding R-loop accumulation and highlights the role of chromatin modifiers and remodelers in facilitating timely R-loop resolution. We also discuss the enigmatic role of RNA:DNA hybrids in facilitating DNA repair, epigenetic landscape and the potential role of replication fork preservation pathways, active fork stability and stalled fork protection pathways, in avoiding replication-transcription conflicts. Finally, we discuss the potential role of several Chro-Mates (chromatin modifiers and remodelers) in the likely differentiation between persistent/detrimental R-loops and transient/benign R-loops that assist in various physiological processes relevant for therapeutic interventions.


Assuntos
Montagem e Desmontagem da Cromatina , Dano ao DNA , Reparo do DNA , Replicação do DNA , Instabilidade Genômica , Estruturas R-Loop , Humanos
4.
DNA Repair (Amst) ; 106: 103182, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34303066

RESUMO

R-loops are non-B DNA structures that form during transcription when the nascent RNA anneals to the template DNA strand forming a RNA:DNA hybrid. Understanding the genomic distribution and function of R-loops is an important goal, since R-loops have been implicated in a number of adaptive and maladaptive processes under physiological and pathological conditions. Based on R-loop mapping datasets, we propose the existence of two main classes of R-loops, each associated with unique characteristics. Promoter-paused R-loops (Class I) are short R-loops that form at high frequency during promoter-proximal pausing by RNA polymerase II. Elongation-associated R-loops (Class II) are long structures that occur throughout gene bodies at modest frequencies. We further discuss the relationships between each R-loop class with instances of genome instability and suggest that increased class I R-loops, resulting from enhanced promoter-proximal pausing, represent the main culprits for R-loop mediated genome instability under pathological conditions.


Assuntos
Instabilidade Genômica , Estruturas R-Loop , Transcrição Genética , Animais , Eucariotos/genética , Eucariotos/metabolismo , Humanos
5.
Nucleic Acids Res ; 49(15): 8573-8591, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34329467

RESUMO

R-loops, which consist of a DNA/RNA hybrid and a displaced single-stranded DNA (ssDNA), are increasingly recognized as critical regulators of chromatin biology. R-loops are particularly enriched at gene promoters, where they play important roles in regulating gene expression. However, the molecular mechanisms that control promoter-associated R-loops remain unclear. The epigenetic 'reader' Tudor domain-containing protein 3 (TDRD3), which recognizes methylarginine marks on histones and on the C-terminal domain of RNA polymerase II, was previously shown to recruit DNA topoisomerase 3B (TOP3B) to relax negatively supercoiled DNA and prevent R-loop formation. Here, we further characterize the function of TDRD3 in R-loop metabolism and introduce the DExH-box helicase 9 (DHX9) as a novel interaction partner of the TDRD3/TOP3B complex. TDRD3 directly interacts with DHX9 via its Tudor domain. This interaction is important for recruiting DHX9 to target gene promoters, where it resolves R-loops in a helicase activity-dependent manner to facilitate gene expression. Additionally, TDRD3 also stimulates the helicase activity of DHX9. This stimulation relies on the OB-fold of TDRD3, which likely binds the ssDNA in the R-loop structure. Thus, DHX9 functions together with TOP3B to suppress promoter-associated R-loops. Collectively, these findings reveal new functions of TDRD3 and provide important mechanistic insights into the regulation of R-loop metabolism.


Assuntos
RNA Helicases DEAD-box/metabolismo , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas , Proteínas/metabolismo , Estruturas R-Loop , Cromatina , DNA Topoisomerases Tipo I/metabolismo , Células HEK293 , Humanos , Células MCF-7 , Domínios e Motivos de Interação entre Proteínas , Proteínas/química , Transcrição Genética
6.
Nat Commun ; 12(1): 4451, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294712

RESUMO

Identifying how R-loops are generated is crucial to know how transcription compromises genome integrity. We show by genome-wide analysis of conditional yeast mutants that the THO transcription complex, prevents R-loop formation in G1 and S-phase, whereas the Sen1 DNA-RNA helicase prevents them only in S-phase. Interestingly, damage accumulates asymmetrically downstream of the replication fork in sen1 cells but symmetrically in the hpr1 THO mutant. Our results indicate that: R-loops form co-transcriptionally independently of DNA replication; that THO is a general and cell-cycle independent safeguard against R-loops, and that Sen1, in contrast to previously believed, is an S-phase-specific R-loop resolvase. These conclusions have important implications for the mechanism of R-loop formation and the role of other factors reported to affect on R-loop homeostasis.


Assuntos
DNA Fúngico/química , Estruturas R-Loop , RNA Fúngico/química , Ciclo Celular/genética , Ciclo Celular/fisiologia , Dano ao DNA , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , Genes Fúngicos , Instabilidade Genômica , Modelos Biológicos , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estruturas R-Loop/genética , Estruturas R-Loop/fisiologia , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
8.
Int J Mol Sci ; 22(12)2021 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-34205418

RESUMO

Endonuclease XPG participates in nucleotide excision repair (NER), in basal transcription, and in the processing of RNA/DNA hybrids (R-loops): the malfunction of these processes may cause genome instability. Here, we investigate the chromatin association of XPG during basal transcription and after transcriptional stress. The inhibition of RNA polymerase II with 5,6-dichloro-l-ß-D-ribofuranosyl benzimidazole (DRB), or actinomycin D (AD), and of topoisomerase I with camptothecin (CPT) resulted in an increase in chromatin-bound XPG, with concomitant relocation by forming nuclear clusters. The cotranscriptional activators p300 and CREB-binding protein (CREBBP), endowed with lysine acetyl transferase (KAT) activity, interact with and acetylate XPG. Depletion of both KATs by RNA interference, or chemical inhibition with C646, significantly reduced XPG acetylation. However, the loss of KAT activity also resulted in increased chromatin association and the relocation of XPG, indicating that these processes were induced by transcriptional stress and not by reduced acetylation. Transcription inhibitors, including C646, triggered the R-loop formation and phosphorylation of histone H2AX (γ-H2AX). Proximity ligation assay (PLA) showed that XPG colocalized with R-loops, indicating the recruitment of the protein to these structures. These results suggest that transcriptional stress-induced XPG relocation may represent recruitment to sites of R-loop processing.


Assuntos
Cromatina/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Linhagem Celular , Histonas/metabolismo , Humanos , Estruturas R-Loop
9.
Nucleic Acids Res ; 49(13): 7618-7627, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34197619

RESUMO

Transcription induced CAG repeat instability is associated with fatal neurological disorders. Genetic approaches found transcription-coupled nucleotide excision repair (TC-NER) factor CSB protein and TFIIS play critical roles in modulating the repeat stability. Here, we took advantage of an in vitro reconstituted yeast transcription system to investigate the underlying mechanism of RNA polymerase II (Pol II) transcriptional pausing/stalling by CAG slip-out structures and the functions of TFIIS and Rad26, the yeast ortholog of CSB, in modulating transcriptional arrest. We identified length-dependent and strand-specific mechanisms that account for CAG slip-out induced transcriptional arrest. We found substantial R-loop formation for the distal transcriptional pausing induced by template strand (TS) slip-out, but not non-template strand (NTS) slip-out. In contrast, Pol II backtracking was observed at the proximal transcriptional pausing sites induced by both NTS and TS slip-out blockage. Strikingly, we revealed that Rad26 and TFIIS can stimulate bypass of NTS CAG slip-out, but not TS slip-out induced distal pausing. Our biochemical results provide new insights into understanding the mechanism of CAG slip-out induced transcriptional pausing and functions of transcription factors in modulating transcription-coupled CAG repeat instability, which may pave the way for developing potential strategies for the treatment of repeat sequence associated human diseases.


Assuntos
Adenosina Trifosfatases/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Elongação da Transcrição Genética , Fatores de Elongação da Transcrição/metabolismo , Repetições de Trinucleotídeos , Estruturas R-Loop
10.
Cell Death Dis ; 12(7): 637, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34155199

RESUMO

Embryonic stem cell (ESC) differentiation and somatic cell reprogramming are biological processes governed by antagonistic expression or repression of a largely common set of genes. Accurate regulation of gene expression is thus essential for both processes, and alterations in RNA processing are predicted to negatively affect both. We show that truncation of the DIDO gene alters RNA splicing and transcription termination in ESC and mouse embryo fibroblasts (MEF), which affects genes involved in both differentiation and reprogramming. We combined transcriptomic, protein interaction, and cellular studies to identify the underlying molecular mechanism. We found that DIDO3 interacts with the helicase DHX9, which is involved in R-loop processing and transcription termination, and that DIDO3-exon16 deletion increases nuclear R-loop content and causes DNA replication stress. Overall, these defects result in failure of ESC to differentiate and of MEF to be reprogrammed. MEF immortalization restored impaired reprogramming capacity. We conclude that DIDO3 has essential functions in ESC differentiation and somatic cell reprogramming by supporting accurate RNA metabolism, with its exon16-encoded domain playing the main role.


Assuntos
Diferenciação Celular , Técnicas de Reprogramação Celular , Reprogramação Celular , Proteínas de Ligação a DNA/genética , Fibroblastos/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Mutação , Estruturas R-Loop , Splicing de RNA , Fatores de Transcrição/genética , Animais , Células Cultivadas , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/patologia , Fenótipo , Fatores de Transcrição/metabolismo , Terminação da Transcrição Genética
11.
Nat Rev Mol Cell Biol ; 22(9): 589-607, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34140671

RESUMO

The human genome contains over one million short tandem repeats. Expansion of a subset of these repeat tracts underlies over fifty human disorders, including common genetic causes of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (C9orf72), polyglutamine-associated ataxias and Huntington disease, myotonic dystrophy, and intellectual disability disorders such as Fragile X syndrome. In this Review, we discuss the four major mechanisms by which expansion of short tandem repeats causes disease: loss of function through transcription repression, RNA-mediated gain of function through gelation and sequestration of RNA-binding proteins, gain of function of canonically translated repeat-harbouring proteins, and repeat-associated non-AUG translation of toxic repeat peptides. Somatic repeat instability amplifies these mechanisms and influences both disease age of onset and tissue specificity of pathogenic features. We focus on the crosstalk between these disease mechanisms, and argue that they often synergize to drive pathogenesis. We also discuss the emerging native functions of repeat elements and how their dynamics might contribute to disease at a larger scale than currently appreciated. Lastly, we propose that lynchpins tying these disease mechanisms and native functions together offer promising therapeutic targets with potential shared applications across this class of human disorders.


Assuntos
Expansão das Repetições de DNA/genética , Doenças Neurodegenerativas/genética , Animais , Inativação Gênica , Instabilidade Genômica , Humanos , Mutação , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/fisiopatologia , Especificidade de Órgãos , Biossíntese de Proteínas , Estruturas R-Loop , RNA/química , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo
12.
Nucleic Acids Res ; 49(13): 7507-7524, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34181717

RESUMO

Impaired replication progression leads to de novo copy number variant (CNV) formation at common fragile sites (CFSs). We previously showed that these hotspots for genome instability reside in late-replicating domains associated with large transcribed genes and provided indirect evidence that transcription is a factor in their instability. Here, we compared aphidicolin (APH)-induced CNV and CFS frequency between wild-type and isogenic cells in which FHIT gene transcription was ablated by promoter deletion. Two promoter-deletion cell lines showed reduced or absent CNV formation and CFS expression at FHIT despite continued instability at the NLGN1 control locus. APH treatment led to critical replication delays that remained unresolved in G2/M in the body of many, but not all, large transcribed genes, an effect that was reversed at FHIT by the promoter deletion. Altering RNase H1 expression did not change CNV induction frequency and DRIP-seq showed a paucity of R-loop formation in the central regions of large genes, suggesting that R-loops are not the primary mediator of the transcription effect. These results demonstrate that large gene transcription is a determining factor in replication stress-induced genomic instability and support models that CNV hotspots mainly result from the transcription-dependent passage of unreplicated DNA into mitosis.


Assuntos
Hidrolases Anidrido Ácido/genética , Variações do Número de Cópias de DNA , Replicação do DNA , Proteínas de Neoplasias/genética , Transcrição Genética , Hidrolases Anidrido Ácido/biossíntese , Animais , Afidicolina/farmacologia , Linhagem Celular , Sítios Frágeis do Cromossomo , Loci Gênicos , Humanos , Camundongos , Mutação , Proteínas de Neoplasias/biossíntese , Regiões Promotoras Genéticas , Estruturas R-Loop , Ribonuclease H/metabolismo , Estresse Fisiológico
13.
Nat Commun ; 12(1): 3686, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140498

RESUMO

Tumour hypoxia is associated with poor patient prognosis and therapy resistance. A unique transcriptional response is initiated by hypoxia which includes the rapid activation of numerous transcription factors in a background of reduced global transcription. Here, we show that the biological response to hypoxia includes the accumulation of R-loops and the induction of the RNA/DNA helicase SETX. In the absence of hypoxia-induced SETX, R-loop levels increase, DNA damage accumulates, and DNA replication rates decrease. Therefore, suggesting that, SETX plays a role in protecting cells from DNA damage induced during transcription in hypoxia. Importantly, we propose that the mechanism of SETX induction in hypoxia is reliant on the PERK/ATF4 arm of the unfolded protein response. These data not only highlight the unique cellular response to hypoxia, which includes both a replication stress-dependent DNA damage response and an unfolded protein response but uncover a novel link between these two distinct pathways.


Assuntos
Hipóxia Celular , Dano ao DNA/genética , DNA Helicases/metabolismo , Regulação da Expressão Gênica/genética , Enzimas Multifuncionais/metabolismo , Estruturas R-Loop/genética , RNA Helicases/metabolismo , Resposta a Proteínas não Dobradas/genética , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Imunoprecipitação da Cromatina , DNA Helicases/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Enzimas Multifuncionais/genética , Inibidores da Síntese de Ácido Nucleico/farmacologia , Oxigênio/farmacologia , Estruturas R-Loop/efeitos dos fármacos , RNA Helicases/genética , RNA-Seq , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Regulação para Cima , Zinostatina/farmacologia , eIF-2 Quinase/metabolismo
14.
Nat Commun ; 12(1): 3849, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158508

RESUMO

DNA-RNA hybrid structures have been detected at the vicinity of DNA double-strand breaks (DSBs) occurring within transcriptional active regions of the genome. The induction of DNA-RNA hybrids strongly affects the repair of these DSBs, but the nature of these structures and how they are formed remain poorly understood. Here we provide evidence that R loops, three-stranded structures containing DNA-RNA hybrids and the displaced single-stranded DNA (ssDNA) can form at sub-telomeric DSBs. These R loops are generated independently of DNA resection but are induced alongside two-stranded DNA-RNA hybrids that form on ssDNA generated by DNA resection. We further identified UPF1, an RNA/DNA helicase, as a crucial factor that drives the formation of these R loops and DNA-RNA hybrids to stimulate DNA resection, homologous recombination, microhomology-mediated end joining and DNA damage checkpoint activation. Our data show that R loops and DNA-RNA hybrids are actively generated at DSBs to facilitate DNA repair.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA/metabolismo , Estruturas R-Loop , RNA Helicases/metabolismo , Transativadores/metabolismo , Sequência de Bases , Linhagem Celular , Linhagem Celular Tumoral , DNA/química , DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Células HCT116 , Humanos , Hibridização de Ácido Nucleico , RNA/genética , RNA/metabolismo , RNA Helicases/genética , Interferência de RNA , Telômero/genética , Telômero/metabolismo , Transativadores/genética
15.
Nat Commun ; 12(1): 3542, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112789

RESUMO

R-loop structures act as modulators of physiological processes such as transcription termination, gene regulation, and DNA repair. However, they can cause transcription-replication conflicts and give rise to genomic instability, particularly at telomeres, which are prone to forming DNA secondary structures. Here, we demonstrate that BRCA1 binds TERRA RNA, directly and physically via its N-terminal nuclear localization sequence, as well as telomere-specific shelterin proteins in an R-loop-, and a cell cycle-dependent manner. R-loop-driven BRCA1 binding to CpG-rich TERRA promoters represses TERRA transcription, prevents TERRA R-loop-associated damage, and promotes its repair, likely in association with SETX and XRN2. BRCA1 depletion upregulates TERRA expression, leading to overly abundant TERRA R-loops, telomeric replication stress, and signs of telomeric aberrancy. Moreover, BRCA1 mutations within the TERRA-binding region lead to an excess of TERRA-associated R-loops and telomeric abnormalities. Thus, normal BRCA1/TERRA binding suppresses telomere-centered genome instability.


Assuntos
Proteína BRCA1/metabolismo , Dano ao DNA/genética , Estruturas R-Loop , RNA Longo não Codificante/metabolismo , Telômero/metabolismo , Proteína BRCA1/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Cromatografia Líquida , Ilhas de CpG , DNA Helicases/metabolismo , Exorribonucleases/metabolismo , Humanos , Hibridização in Situ Fluorescente , Espectrometria de Massas , Enzimas Multifuncionais/metabolismo , Mutação , Regiões Promotoras Genéticas , Ligação Proteica , Estruturas R-Loop/genética , RNA Helicases/metabolismo , RNA Longo não Codificante/genética , RNA Interferente Pequeno , Telômero/genética
16.
Nat Commun ; 12(1): 3476, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108490

RESUMO

Cas12i is a newly identified member of the functionally diverse type V CRISPR-Cas effectors. Although Cas12i has the potential to serve as genome-editing tool, its structural and functional characteristics need to be investigated in more detail before effective application. Here we report the crystal structures of the Cas12i1 R-loop complexes before and after target DNA cleavage to elucidate the mechanisms underlying target DNA duplex unwinding, R-loop formation and cis cleavage. The structure of the R-loop complex after target DNA cleavage also provides information regarding trans cleavage. Besides, we report a crystal structure of the Cas12i1 binary complex interacting with a pseudo target oligonucleotide, which mimics target interrogation. Upon target DNA duplex binding, the Cas12i1 PAM-interacting cleft undergoes a remarkable open-to-closed adjustment. Notably, a zipper motif in the Helical-I domain facilitates unzipping of the target DNA duplex. Formation of the 19-bp crRNA-target DNA strand heteroduplex in the R-loop complexes triggers a conformational rearrangement and unleashes the DNase activity. This study provides valuable insights for developing Cas12i1 into a reliable genome-editing tool.


Assuntos
Proteínas Associadas a CRISPR/química , Clivagem do DNA , Endonucleases/química , Estruturas R-Loop , Pareamento de Bases , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Domínio Catalítico , DNA/química , DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Ativação Enzimática , Magnésio/química , Modelos Biológicos , Modelos Moleculares , Conformação Proteica , Estrutura Terciária de Proteína , RNA Guia/química , RNA Guia/metabolismo , Temperatura
17.
DNA Repair (Amst) ; 104: 103132, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34049076

RESUMO

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.


Assuntos
Dano ao DNA , Replicação do DNA , Instabilidade Genômica , Poli(ADP-Ribose) Polimerase-1/metabolismo , Estruturas R-Loop , Fatores de Transcrição/metabolismo , Transcrição Genética , Camptotecina/toxicidade , Linhagem Celular , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Poli ADP Ribosilação
18.
Nat Commun ; 12(1): 3016, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34021146

RESUMO

Telomere repeat containing RNAs (TERRAs) are a family of long non-coding RNAs transcribed from the subtelomeric regions of eukaryotic chromosomes. TERRA transcripts can form R-loops at chromosome ends; however the importance of these structures or the regulation of TERRA expression and retention in telomeric R-loops remain unclear. Here, we show that the RTEL1 (Regulator of Telomere Length 1) helicase influences the abundance and localization of TERRA in human cells. Depletion of RTEL1 leads to increased levels of TERRA RNA while reducing TERRA-containing R loops at telomeres. In vitro, RTEL1 shows a strong preference for binding G-quadruplex structures which form in TERRA. This binding is mediated by the C-terminal region of RTEL1, and is independent of the RTEL1 helicase domain. RTEL1 binding to TERRA appears to be essential for cell viability, underscoring the importance of this function. Degradation of TERRA-containing R-loops by overexpression of RNAse H1 partially recapitulates the increased TERRA levels and telomeric instability associated with RTEL1 deficiency. Collectively, these data suggest that regulation of TERRA is a key function of the RTEL1 helicase, and that loss of that function may contribute to the disease phenotypes of patients with RTEL1 mutations.


Assuntos
DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , DNA Helicases/química , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Domínios Proteicos , Estruturas R-Loop , RNA , Ribonuclease H , Alinhamento de Sequência , Telômero , Fatores de Transcrição/genética
19.
Nat Commun ; 12(1): 3153, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34039990

RESUMO

RNA splicing, transcription and the DNA damage response are intriguingly linked in mammals but the underlying mechanisms remain poorly understood. Using an in vivo biotinylation tagging approach in mice, we show that the splicing factor XAB2 interacts with the core spliceosome and that it binds to spliceosomal U4 and U6 snRNAs and pre-mRNAs in developing livers. XAB2 depletion leads to aberrant intron retention, R-loop formation and DNA damage in cells. Studies in illudin S-treated cells and Csbm/m developing livers reveal that transcription-blocking DNA lesions trigger the release of XAB2 from all RNA targets tested. Immunoprecipitation studies reveal that XAB2 interacts with ERCC1-XPF and XPG endonucleases outside nucleotide excision repair and that the trimeric protein complex binds RNA:DNA hybrids under conditions that favor the formation of R-loops. Thus, XAB2 functionally links the spliceosomal response to DNA damage with R-loop processing with important ramifications for transcription-coupled DNA repair disorders.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Processamento de RNA/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Dano ao DNA/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Introdução de Genes , Técnicas de Silenciamento de Genes , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas , Sesquiterpenos Policíclicos/farmacologia , Estruturas R-Loop/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Fatores de Processamento de RNA/genética , RNA Nuclear Pequeno , RNA-Seq , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spliceossomos/metabolismo , Transcrição Genética
20.
Nature ; 594(7862): 283-288, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33981036

RESUMO

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 protein 1 (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.


Assuntos
DNA/genética , DNA/metabolismo , Recombinação Homóloga/genética , Estruturas R-Loop/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular , DNA/química , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Genes/genética , Genes Reporter/genética , Proteínas de Fluorescência Verde/genética , Humanos , Técnicas In Vitro , RNA Mensageiro/química , Proteínas de Ligação a RNA/metabolismo , Rad51 Recombinase/metabolismo
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