RESUMO
DdmDE is a novel plasmid defense system that was discovered in the seventh pandemic Vibrio cholerae strain of the biotype O1 EI Tor. In this issue of Cell, Yang and coworkers reveal the mechanisms underlying the assembly and activation of the DdmDE defense system.
Assuntos
Plasmídeos , Vibrio cholerae , Plasmídeos/metabolismo , Plasmídeos/genética , Vibrio cholerae/genética , Vibrio cholerae/metabolismo , Vibrio cholerae/enzimologia , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , DNA Helicases/metabolismo , DNA Helicases/genéticaRESUMO
Homologous recombination (HR) helps maintain genome integrity, and HR defects give rise to disease, especially cancer. During HR, damaged DNA must be aligned with an undamaged template through a process referred to as the homology search. Despite decades of study, key aspects of this search remain undefined. Here, we use single-molecule imaging to demonstrate that Rad54, a conserved Snf2-like protein found in all eukaryotes, switches the search from the diffusion-based pathways characteristic of the basal HR machinery to an active process in which DNA sequences are aligned via an ATP-dependent molecular motor-driven mechanism. We further demonstrate that Rad54 disrupts the donor template strands, enabling the search to take place within a migrating DNA bubble-like structure that is bound by replication protein A (RPA). Our results reveal that Rad54, working together with RPA, fundamentally alters how DNA sequences are aligned during HR.
Assuntos
Trifosfato de Adenosina/genética , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , DNA/genética , Recombinação Homóloga/genética , Proteínas de Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/genética , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Hidrólise , Saccharomyces cerevisiae/genética , Alinhamento de Sequência/métodosRESUMO
The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity, and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 31 CRISPR-Cas9 screens against 27 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 890 genes whose loss causes either sensitivity or resistance to DNA-damaging agents. Mining this dataset, we discovered that ERCC6L2 (which is mutated in a bone-marrow failure syndrome) codes for a canonical non-homologous end-joining pathway factor, that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents, and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.
Assuntos
Dano ao DNA , Redes Reguladoras de Genes/fisiologia , Aminoquinolinas/farmacologia , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular , Citocromo-B(5) Redutase/genética , Citocromo-B(5) Redutase/metabolismo , Dano ao DNA/efeitos dos fármacos , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Humanos , Camundongos , Ácidos Picolínicos/farmacologia , RNA Guia de Cinetoplastídeos/metabolismo , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
Mammalian SWI/SNF complexes are ATP-dependent chromatin remodeling complexes that regulate genomic architecture. Here, we present a structural model of the endogenously purified human canonical BAF complex bound to the nucleosome, generated using cryoelectron microscopy (cryo-EM), cross-linking mass spectrometry, and homology modeling. BAF complexes bilaterally engage the nucleosome H2A/H2B acidic patch regions through the SMARCB1 C-terminal α-helix and the SMARCA4/2 C-terminal SnAc/post-SnAc regions, with disease-associated mutations in either causing attenuated chromatin remodeling activities. Further, we define changes in BAF complex architecture upon nucleosome engagement and compare the structural model of endogenous BAF to those of related SWI/SNF-family complexes. Finally, we assign and experimentally interrogate cancer-associated hot-spot mutations localizing within the endogenous human BAF complex, identifying those that disrupt BAF subunit-subunit and subunit-nucleosome interfaces in the nucleosome-bound conformation. Taken together, this integrative structural approach provides important biophysical foundations for understanding the mechanisms of BAF complex function in normal and disease states.
Assuntos
Doença , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Montagem e Desmontagem da Cromatina , Microscopia Crioeletrônica , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/metabolismo , Doença/genética , Humanos , Mutação de Sentido Incorreto/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Ligação Proteica , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Homologia Estrutural de Proteína , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Telomere maintenance critically depends on the distinct activities of telomerase, which adds telomeric repeats to solve the end replication problem, and RTEL1, which dismantles DNA secondary structures at telomeres to facilitate replisome progression. Here, we establish that reversed replication forks are a pathological substrate for telomerase and the source of telomere catastrophe in Rtel1-/- cells. Inhibiting telomerase recruitment to telomeres, but not its activity, or blocking replication fork reversal through PARP1 inhibition or depleting UBC13 or ZRANB3 prevents the rapid accumulation of dysfunctional telomeres in RTEL1-deficient cells. In this context, we establish that telomerase binding to reversed replication forks inhibits telomere replication, which can be mimicked by preventing replication fork restart through depletion of RECQ1 or PARG. Our results lead us to propose that telomerase inappropriately binds to and inhibits restart of reversed replication forks within telomeres, which compromises replication and leads to critically short telomeres.
Assuntos
DNA Helicases/genética , Replicação do DNA , Homeostase do Telômero , Animais , Linhagem Celular , Células Cultivadas , DNA Helicases/metabolismo , Glicosídeo Hidrolases/metabolismo , Camundongos , Poli(ADP-Ribose) Polimerase-1/metabolismo , RecQ Helicases/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.
Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , Animais , Linhagem Celular Tumoral , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Metilação de DNA , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
This review focuses on the biogenesis and composition of the eukaryotic DNA replication fork, with an emphasis on the enzymes that synthesize DNA and repair discontinuities on the lagging strand of the replication fork. Physical and genetic methodologies aimed at understanding these processes are discussed. The preponderance of evidence supports a model in which DNA polymerase ε (Pol ε) carries out the bulk of leading strand DNA synthesis at an undisturbed replication fork. DNA polymerases α and δ carry out the initiation of Okazaki fragment synthesis and its elongation and maturation, respectively. This review also discusses alternative proposals, including cellular processes during which alternative forks may be utilized, and new biochemical studies with purified proteins that are aimed at reconstituting leading and lagging strand DNA synthesis separately and as an integrated replication fork.
Assuntos
DNA Helicases/genética , DNA Polimerase II/genética , Replicação do DNA , DNA/genética , Células Eucarióticas/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA/metabolismo , DNA Helicases/metabolismo , DNA Polimerase I/genética , DNA Polimerase I/metabolismo , DNA Polimerase II/metabolismo , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Eucarióticas/citologia , Humanos , Proteínas de Manutenção de Minicromossomo/genética , Proteínas de Manutenção de Minicromossomo/metabolismoRESUMO
The transcription-related DNA damage response was analyzed on a genome-wide scale with great spatial and temporal resolution. Upon UV irradiation, a slowdown of transcript elongation and restriction of gene activity to the promoter-proximal â¼25 kb is observed. This is associated with a shift from expression of long mRNAs to shorter isoforms, incorporating alternative last exons (ALEs) that are more proximal to the transcription start site. Notably, this includes a shift from a protein-coding ASCC3 mRNA to a shorter ALE isoform of which the RNA, rather than an encoded protein, is critical for the eventual recovery of transcription. The non-coding ASCC3 isoform counteracts the function of the protein-coding isoform, indicating crosstalk between them. Thus, the ASCC3 gene expresses both coding and non-coding transcript isoforms with opposite effects on transcription recovery after UV-induced DNA damage.
Assuntos
Processamento Alternativo/efeitos da radiação , DNA Helicases/genética , RNA não Traduzido/genética , Transcrição Gênica , Raios Ultravioleta , Linhagem Celular , Éxons , Humanos , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elongação da Transcrição Genética/efeitos da radiação , Iniciação da Transcrição Genética/efeitos da radiaçãoRESUMO
DNA polymerase θ (Polθ) plays a central role in a DNA double-strand break repair pathway termed theta-mediated end joining (TMEJ). TMEJ functions by pairing short-sequence "microhomologies" (MHs) in single-stranded DNA at each end of a break and subsequently initiating DNA synthesis. It is not known how the Polθ helicase domain (HD) and polymerase domain (PD) operate to bring together MHs and facilitate repair. To resolve these transient processes in real time, we utilized in vitro single-molecule FRET approaches and biochemical analyses. We find that the Polθ-HD mediates the initial capture of two ssDNA strands, bringing them in close proximity. The Polθ-PD binds and stabilizes pre-annealed MHs to form a synaptic complex (SC) and initiate repair synthesis. Individual synthesis reactions show that Polθ is inherently non-processive, accounting for complex mutational patterns during TMEJ. Binding of Polθ-PD to stem-loop-forming sequences can substantially limit synapsis, depending on the available dNTPs and sequence context.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Polimerase Dirigida por DNA , DNA Polimerase Dirigida por DNA/metabolismo , Replicação do DNA , DNA de Cadeia Simples/genética , DNA Helicases/genética , Reparo do DNA por Junção de ExtremidadesRESUMO
RNA transcribed from enhancers, i.e., eRNA, has been suggested to directly activate transcription by recruiting transcription factors and co-activators. Although there have been specific examples of eRNA functioning in this way, it is not clear how general this may be. We find that the AT-hook of SWI/SNF preferentially binds RNA and, as part of the esBAF complex, associates with eRNA transcribed from intronic and intergenic regions. Our data suggest that SWI/SNF is globally recruited in cis by eRNA to cell-type-specific enhancers, representative of two distinct stages that mimic early mammalian development, and not at enhancers that are shared between the two stages. In this manner, SWI/SNF facilitates recruitment and/or activation of MLL3/4, p300/CBP, and Mediator to stage-specific enhancers and super-enhancers that regulate the transcription of metabolic and cell lineage priming-related genes. These findings highlight a connection between ATP-dependent chromatin remodeling and eRNA in cell identity and typical- and super-enhancer activation.
Assuntos
Linhagem da Célula , DNA Helicases , Elementos Facilitadores Genéticos , Proteínas Nucleares , Fatores de Transcrição , Animais , Humanos , Linhagem da Célula/genética , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , DNA Helicases/metabolismo , DNA Helicases/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
In this issue of Molecular Cell, Engeholm et al.1 present cryo-EM structures of the chromatin remodeler Chd1 bound to a hexasome-nucleosome complex, an intermediate state during transcription either with or without FACT to restore the missing H2A-H2B dimer. These two binding modes reveal how Chd1 and FACT cooperate in nucleosome re-establishment during transcription.
Assuntos
Microscopia Crioeletrônica , Proteínas de Ligação a DNA , Nucleossomos , Nucleossomos/metabolismo , Nucleossomos/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Montagem e Desmontagem da Cromatina , Histonas/metabolismo , Histonas/genética , Humanos , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , DNA Helicases/metabolismo , DNA Helicases/genética , Ligação Proteica , Transcrição Gênica , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Grupo de Alta Mobilidade/genética , Fatores de Elongação da Transcrição/metabolismo , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/químicaRESUMO
The Bloom syndrome (BLM) helicase is critical for alternative lengthening of telomeres (ALT), a homology-directed repair (HDR)-mediated telomere maintenance mechanism that is prevalent in cancers of mesenchymal origin. The DNA substrates that BLM engages to direct telomere recombination during ALT remain unknown. Here, we determine that BLM helicase acts on lagging strand telomere intermediates that occur specifically in ALT-positive cells to assemble a replication-associated DNA damage response. Loss of ATRX was permissive for BLM localization to ALT telomeres in S and G2, commensurate with the appearance of telomere C-strand-specific single-stranded DNA (ssDNA). DNA2 nuclease deficiency increased 5'-flap formation in a BLM-dependent manner, while telomere C-strand, but not G-strand, nicks promoted ALT. These findings define the seminal events in the ALT DNA damage response, linking aberrant telomeric lagging strand DNA replication with a BLM-directed HDR mechanism that sustains telomere length in a subset of human cancers.
Assuntos
Dano ao DNA , Replicação do DNA , RecQ Helicases , Homeostase do Telômero , Telômero , RecQ Helicases/metabolismo , RecQ Helicases/genética , Humanos , Telômero/metabolismo , Telômero/genética , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Proteína Nuclear Ligada ao X/genética , Proteína Nuclear Ligada ao X/metabolismo , DNA Helicases/metabolismo , DNA Helicases/genética , Síndrome de Bloom/genética , Síndrome de Bloom/metabolismo , Síndrome de Bloom/enzimologia , Síndrome de Bloom/patologia , Linhagem Celular TumoralRESUMO
Long noncoding (lnc)RNAs emerge as regulators of genome stability. The nuclear-enriched abundant transcript 1 (NEAT1) is overexpressed in many tumors and is responsive to genotoxic stress. However, the mechanism that links NEAT1 to DNA damage response (DDR) is unclear. Here, we investigate the expression, modification, localization, and structure of NEAT1 in response to DNA double-strand breaks (DSBs). DNA damage increases the levels and N6-methyladenosine (m6A) marks on NEAT1, which promotes alterations in NEAT1 structure, accumulation of hypermethylated NEAT1 at promoter-associated DSBs, and DSB signaling. The depletion of NEAT1 impairs DSB focus formation and elevates DNA damage. The genome-protective role of NEAT1 is mediated by the RNA methyltransferase 3 (METTL3) and involves the release of the chromodomain helicase DNA binding protein 4 (CHD4) from NEAT1 to fine-tune histone acetylation at DSBs. Our data suggest a direct role for NEAT1 in DDR.
Assuntos
Adenosina , Quebras de DNA de Cadeia Dupla , Instabilidade Genômica , Metiltransferases , RNA Longo não Codificante , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Instabilidade Genômica/genética , Humanos , Adenosina/análogos & derivados , Adenosina/metabolismo , Metiltransferases/metabolismo , Metiltransferases/genética , Metilação , Dano ao DNA/genética , DNA Helicases/metabolismo , DNA Helicases/genética , Histonas/metabolismo , Histonas/genética , Regulação da Expressão GênicaRESUMO
Cyclic GMP-AMP synthase (cGAS) is a key sensor responsible for cytosolic DNA detection. Here we report that GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) is critical for DNA sensing and efficient activation of cGAS. G3BP1 enhanced DNA binding of cGAS by promoting the formation of large cGAS complexes. G3BP1 deficiency led to inefficient DNA binding by cGAS and inhibited cGAS-dependent interferon (IFN) production. The G3BP1 inhibitor epigallocatechin gallate (EGCG) disrupted existing G3BP1-cGAS complexes and inhibited DNA-triggered cGAS activation, thereby blocking DNA-induced IFN production both in vivo and in vitro. EGCG administration blunted self DNA-induced autoinflammatory responses in an Aicardi-Goutières syndrome (AGS) mouse model and reduced IFN-stimulated gene expression in cells from a patient with AGS. Thus, our study reveals that G3BP1 physically interacts with and primes cGAS for efficient activation. Furthermore, EGCG-mediated inhibition of G3BP1 provides a potential treatment for cGAS-related autoimmune diseases.
Assuntos
Doenças Autoimunes do Sistema Nervoso/metabolismo , DNA Helicases/metabolismo , Complexos Multiproteicos/metabolismo , Malformações do Sistema Nervoso/metabolismo , Nucleotidiltransferases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Animais , Autoantígenos/imunologia , Autoantígenos/metabolismo , Doenças Autoimunes do Sistema Nervoso/tratamento farmacológico , Doenças Autoimunes do Sistema Nervoso/genética , Catequina/análogos & derivados , Catequina/uso terapêutico , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Citosol/imunologia , Citosol/metabolismo , DNA/imunologia , DNA/metabolismo , DNA Helicases/antagonistas & inibidores , DNA Helicases/genética , Modelos Animais de Doenças , Exodesoxirribonucleases/genética , Células HEK293 , Células HeLa , Humanos , Interferons/metabolismo , Camundongos , Camundongos Knockout , Malformações do Sistema Nervoso/tratamento farmacológico , Malformações do Sistema Nervoso/genética , Fármacos Neuroprotetores/uso terapêutico , Fosfoproteínas/genética , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Proteínas de Ligação a Poli-ADP-Ribose/genética , Ligação Proteica , RNA Helicases/antagonistas & inibidores , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/antagonistas & inibidores , Proteínas com Motivo de Reconhecimento de RNA/genéticaRESUMO
Therapy development for adult diffuse glioma is hindered by incomplete knowledge of somatic glioma driving alterations and suboptimal disease classification. We defined the complete set of genes associated with 1,122 diffuse grade II-III-IV gliomas from The Cancer Genome Atlas and used molecular profiles to improve disease classification, identify molecular correlations, and provide insights into the progression from low- to high-grade disease. Whole-genome sequencing data analysis determined that ATRX but not TERT promoter mutations are associated with increased telomere length. Recent advances in glioma classification based on IDH mutation and 1p/19q co-deletion status were recapitulated through analysis of DNA methylation profiles, which identified clinically relevant molecular subsets. A subtype of IDH mutant glioma was associated with DNA demethylation and poor outcome; a group of IDH-wild-type diffuse glioma showed molecular similarity to pilocytic astrocytoma and relatively favorable survival. Understanding of cohesive disease groups may aid improved clinical outcomes.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Glioma/genética , Glioma/patologia , Transcriptoma , Adulto , Neoplasias Encefálicas/metabolismo , Proliferação de Células , Análise por Conglomerados , DNA Helicases/genética , Metilação de DNA , Epigênese Genética , Glioma/metabolismo , Humanos , Isocitrato Desidrogenase/genética , Pessoa de Meia-Idade , Mutação , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Transdução de Sinais , Telomerase/genética , Telômero , Proteína Nuclear Ligada ao XRESUMO
To investigate epigenetic dependencies and identify therapeutic vulnerabilities, Mo et al.1 and Panditharatna et al.2 performed CRISPR screens and show that deadly H3K27M gliomas are dependent on mammalian BAF (SWI/SNF) chromatin remodeling complex.
Assuntos
Glioma , Animais , Humanos , Glioma/genética , Mamíferos , DNA Helicases/genética , Proteínas Nucleares , Fatores de TranscriçãoRESUMO
Cells must avoid licensing of neosynthesized DNA to prevent rereplication. In this issue of Molecular Cell, Ratnayeke et al. (2022)1 reveal how the licensing factor CDT1, prior to its degradation, inhibits DNA elongation by suppressing CMG helicase progression at replication forks.
Assuntos
Proteínas de Ciclo Celular , Replicação do DNA , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/genética , DNA , DNA Helicases/genéticaRESUMO
The senataxin (SETX, Sen1 in yeasts) RNA-DNA hybrid resolving helicase regulates multiple nuclear transactions, including DNA replication, transcription, and DNA repair, but the molecular basis for Sen1 activities is ill defined. Here, Sen1 cryoelectron microscopy (cryo-EM) reconstructions reveal an elongated inchworm-like architecture. Sen1 is composed of an amino terminal helical repeat Sen1 N-terminal (Sen1N) regulatory domain that is flexibly linked to its C-terminal SF1B helicase motor core (Sen1Hel) via an intrinsically disordered tether. In an autoinhibited state, the Sen1Sen1N domain regulates substrate engagement by promoting occlusion of the RNA substrate-binding cleft. The X-ray structure of an activated Sen1Hel engaging single-stranded RNA and ADP-SO4 shows that the enzyme encircles RNA and implicates a single-nucleotide power stroke in the Sen1 RNA translocation mechanism. Together, our data unveil dynamic protein-protein and protein-RNA interfaces underpinning helicase regulation and inactivation of human SETX activity by RNA-binding-deficient mutants in ataxia with oculomotor apraxia 2 neurodegenerative disease.
Assuntos
Doenças Neurodegenerativas , RNA , Humanos , RNA/genética , Microscopia Crioeletrônica , RNA Helicases/genética , RNA Helicases/química , Enzimas Multifuncionais/genética , DNA/genética , Homeostase , DNA Helicases/genéticaRESUMO
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.
Assuntos
Estruturas R-Loop , Ribonucleases , Humanos , Estruturas R-Loop/genética , Ribonucleases/genética , RNA/genética , DNA , Replicação do DNA , DNA Helicases/genética , Ribonuclease H/genética , Ribonuclease H/metabolismo , Instabilidade GenômicaRESUMO
DNA replication preferentially initiates close to active transcription start sites (TSSs) in the human genome. Transcription proceeds discontinuously with an accumulation of RNA polymerase II (RNAPII) in a paused state near the TSS. Consequently, replication forks inevitably encounter paused RNAPII soon after replication initiates. Hence, dedicated machinery may be needed to remove RNAPII and facilitate unperturbed fork progression. In this study, we discovered that Integrator, a transcription termination machinery involved in the processing of RNAPII transcripts, interacts with the replicative helicase at active forks and promotes the removal of RNAPII from the path of the replication fork. Integrator-deficient cells have impaired replication fork progression and accumulate hallmarks of genome instability including chromosome breaks and micronuclei. The Integrator complex resolves co-directional transcription-replication conflicts to facilitate faithful DNA replication.