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1.
Mol Cell ; 83(13): 2258-2275.e11, 2023 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-37369199

RESUMO

The pre-mRNA life cycle requires intron processing; yet, how intron-processing defects influence splicing and gene expression is unclear. Here, we find that TTDN1/MPLKIP, which is encoded by a gene implicated in non-photosensitive trichothiodystrophy (NP-TTD), functionally links intron lariat processing to spliceosomal function. The conserved TTDN1 C-terminal region directly binds lariat debranching enzyme DBR1, whereas its N-terminal intrinsically disordered region (IDR) binds the intron-binding complex (IBC). TTDN1 loss, or a mutated IDR, causes significant intron lariat accumulation, as well as splicing and gene expression defects, mirroring phenotypes observed in NP-TTD patient cells. A Ttdn1-deficient mouse model recapitulates intron-processing defects and certain neurodevelopmental phenotypes seen in NP-TTD. Fusing DBR1 to the TTDN1 IDR is sufficient to recruit DBR1 to the IBC and circumvents the functional requirement for TTDN1. Collectively, our findings link RNA lariat processing with splicing outcomes by revealing the molecular function of TTDN1.


Assuntos
Síndromes de Tricotiodistrofia , Animais , Camundongos , Íntrons/genética , Síndromes de Tricotiodistrofia/genética , RNA Nucleotidiltransferases/genética , Splicing de RNA
2.
Mol Cell ; 82(12): 2267-2297, 2022 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-35508167

RESUMO

Although transcription is an essential cellular process, it is paradoxically also a well-recognized cause of genomic instability. R-loops, non-B DNA structures formed when nascent RNA hybridizes to DNA to displace the non-template strand as single-stranded DNA (ssDNA), are partially responsible for this instability. Yet, recent work has begun to elucidate regulatory roles for R-loops in maintaining the genome. In this review, we discuss the cellular contexts in which R-loops contribute to genomic instability, particularly during DNA replication and double-strand break (DSB) repair. We also summarize the evidence that R-loops participate as an intermediate during repair and may influence pathway choice to preserve genomic integrity. Finally, we discuss the immunogenic potential of R-loops and highlight their links to disease should they become pathogenic.


Assuntos
Estruturas R-Loop , Transcrição Gênica , DNA/metabolismo , Reparo do DNA , Replicação do DNA , DNA de Cadeia Simples/genética , Instabilidade Genômica , Humanos , Estruturas R-Loop/genética
3.
Nature ; 613(7942): 187-194, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36544021

RESUMO

R-loops are RNA-DNA-hybrid-containing nucleic acids with important cellular roles. Deregulation of R-loop dynamics can lead to DNA damage and genome instability1, which has been linked to the action of endonucleases such as XPG2-4. However, the mechanisms and cellular consequences of such processing have remained unclear. Here we identify a new population of RNA-DNA hybrids in the cytoplasm that are R-loop-processing products. When nuclear R-loops were perturbed by depleting the RNA-DNA helicase senataxin (SETX) or the breast cancer gene BRCA1 (refs. 5-7), we observed XPG- and XPF-dependent cytoplasmic hybrid formation. We identify their source as a subset of stable, overlapping nuclear hybrids with a specific nucleotide signature. Cytoplasmic hybrids bind to the pattern recognition receptors cGAS and TLR3 (ref. 8), activating IRF3 and inducing apoptosis. Excised hybrids and an R-loop-induced innate immune response were also observed in SETX-mutated cells from patients with ataxia oculomotor apraxia type 2 (ref. 9) and in BRCA1-mutated cancer cells10. These findings establish RNA-DNA hybrids as immunogenic species that aberrantly accumulate in the cytoplasm after R-loop processing, linking R-loop accumulation to cell death through the innate immune response. Aberrant R-loop processing and subsequent innate immune activation may contribute to many diseases, such as neurodegeneration and cancer.


Assuntos
Citoplasma , DNA , Reconhecimento da Imunidade Inata , Ácidos Nucleicos Heteroduplexes , Estruturas R-Loop , RNA , Humanos , Apoptose , Citoplasma/imunologia , Citoplasma/metabolismo , DNA/química , DNA/imunologia , DNA Helicases/genética , DNA Helicases/metabolismo , Genes BRCA1 , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Mutação , Neoplasias , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Heteroduplexes/imunologia , Estruturas R-Loop/imunologia , RNA/química , RNA/imunologia , RNA Helicases/genética , RNA Helicases/metabolismo , Ataxias Espinocerebelares/genética
4.
Mol Cell ; 81(20): 4228-4242.e8, 2021 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-34686315

RESUMO

Central to genotoxic responses is their ability to sense highly specific signals to activate the appropriate repair response. We previously reported that the activation of the ASCC-ALKBH3 repair pathway is exquisitely specific to alkylation damage in human cells. Yet the mechanistic basis for the selectivity of this pathway was not immediately obvious. Here, we demonstrate that RNA but not DNA alkylation is the initiating signal for this process. Aberrantly methylated RNA is sufficient to recruit ASCC, while an RNA dealkylase suppresses ASCC recruitment during chemical alkylation. In turn, recruitment of ASCC during alkylation damage, which is mediated by the E3 ubiquitin ligase RNF113A, suppresses transcription and R-loop formation. We further show that alkylated pre-mRNA is sufficient to activate RNF113A E3 ligase in vitro in a manner dependent on its RNA binding Zn-finger domain. Together, our work identifies an unexpected role for RNA damage in eliciting a specific response to genotoxins.


Assuntos
Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Núcleo Celular/enzimologia , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias/enzimologia , Proteínas Nucleares/metabolismo , Processamento Pós-Transcricional do RNA , RNA Neoplásico/metabolismo , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/genética , Núcleo Celular/genética , DNA Helicases/genética , Metilação de DNA , Proteínas de Ligação a DNA/genética , Células HEK293 , Células HeLa , Humanos , Metilação , Neoplasias/genética , Proteínas Nucleares/genética , Estruturas R-Loop , RNA Neoplásico/genética , Spliceossomos/genética , Spliceossomos/metabolismo , Transcrição Gênica , Ubiquitinação
6.
Nature ; 551(7680): 389-393, 2017 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-29144457

RESUMO

DNA repair is essential to prevent the cytotoxic or mutagenic effects of various types of DNA lesions, which are sensed by distinct pathways to recruit repair factors specific to the damage type. Although biochemical mechanisms for repairing several forms of genomic insults are well understood, the upstream signalling pathways that trigger repair are established for only certain types of damage, such as double-stranded breaks and interstrand crosslinks. Understanding the upstream signalling events that mediate recognition and repair of DNA alkylation damage is particularly important, since alkylation chemotherapy is one of the most widely used systemic modalities for cancer treatment and because environmental chemicals may trigger DNA alkylation. Here we demonstrate that human cells have a previously unrecognized signalling mechanism for sensing damage induced by alkylation. We find that the alkylation repair complex ASCC (activating signal cointegrator complex) relocalizes to distinct nuclear foci specifically upon exposure of cells to alkylating agents. These foci associate with alkylated nucleotides, and coincide spatially with elongating RNA polymerase II and splicing components. Proper recruitment of the repair complex requires recognition of K63-linked polyubiquitin by the CUE (coupling of ubiquitin conjugation to ER degradation) domain of the subunit ASCC2. Loss of this subunit impedes alkylation adduct repair kinetics and increases sensitivity to alkylating agents, but not other forms of DNA damage. We identify RING finger protein 113A (RNF113A) as the E3 ligase responsible for upstream ubiquitin signalling in the ASCC pathway. Cells from patients with X-linked trichothiodystrophy, which harbour a mutation in RNF113A, are defective in ASCC foci formation and are hypersensitive to alkylating agents. Together, our work reveals a previously unrecognized ubiquitin-dependent pathway induced specifically to repair alkylation damage, shedding light on the molecular mechanism of X-linked trichothiodystrophy.


Assuntos
Enzimas AlkB/metabolismo , Adutos de DNA/metabolismo , Reparo do DNA , Complexos Multiproteicos/metabolismo , Transdução de Sinais , Síndromes de Tricotiodistrofia/genética , Ubiquitina/metabolismo , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Alquilantes/farmacologia , Alquilação , Sequência de Aminoácidos , Adutos de DNA/química , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Retículo Endoplasmático/metabolismo , Genes Ligados ao Cromossomo X , Humanos , Cinética , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Poliubiquitina/metabolismo , RNA Polimerase II/metabolismo , Splicing de RNA , Síndromes de Tricotiodistrofia/metabolismo , Síndromes de Tricotiodistrofia/patologia , Ubiquitinação
7.
Biochemistry ; 58(5): 312-329, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30346748

RESUMO

An emerging molecular understanding of RNA alkylation and its removal is transforming our knowledge of RNA biology and its interplay with cancer chemotherapy responses. DNA modifications are known to perform critical functions depending on the genome template, including gene expression, DNA replication timing, and DNA damage protection, yet current results suggest that the chemical diversity of DNA modifications pales in comparison to those on RNA. More than 150 RNA modifications have been identified to date, and their complete functional implications are still being unveiled. These include intrinsic roles such as proper processing and RNA maturation; emerging evidence has furthermore uncovered RNA modification "readers", seemingly analogous to those identified for histone modifications. These modification recognition factors may regulate mRNA stability, localization, and interaction with translation machinery, affecting gene expression. Not surprisingly, tumors differentially modulate factors involved in expressing these marks, contributing to both tumorigenesis and responses to alkylating chemotherapy. Here we describe the current understanding of RNA modifications and their removal, with a focus primarily on methylation and alkylation as functionally relevant changes to the transcriptome. Intriguingly, some of the same RNA modifications elicited by physiological processes are also produced by alkylating agents, thus blurring the lines between what is a physiological mark and a damage-induced modification. Furthermore, we find that a high level of gene expression of enzymes with RNA dealkylation activity is a sensitive readout for poor survival in four different cancer types, underscoring the likely importance of examining RNA dealkylation mechanisms to cancer biology and for cancer treatment and prognosis.


Assuntos
Epigênese Genética , Neoplasias/patologia , Processamento Pós-Transcricional do RNA , RNA/química , RNA/genética , Alquilação , Humanos , Metilação , Neoplasias/genética
8.
J Biol Chem ; 293(35): 13524-13533, 2018 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-29997253

RESUMO

Multiple DNA damage response (DDR) pathways have evolved to sense the presence of damage and recruit the proper repair factors. We recently reported a signaling pathway induced upon alkylation damage to recruit the AlkB homolog 3, α-ketoglutarate-dependent dioxygenase (ALKBH3)-activating signal cointegrator 1 complex subunit 3 (ASCC3) dealkylase-helicase repair complex. As in other DDR pathways, the recruitment of these repair factors is mediated through a ubiquitin-dependent mechanism. However, the machinery that coordinates the proper assembly of this repair complex and controls its recruitment is still poorly defined. Here, we demonstrate that the ASCC1 accessory subunit is important for the regulation of ASCC complex function. ASCC1 interacts with the ASCC complex through the ASCC3 helicase subunit. We find that ASCC1 is present at nuclear speckle foci prior to damage, but leaves the foci in response to alkylation. Strikingly, ASCC1 loss significantly increases ASCC3 foci formation during alkylation damage, yet most of these foci lack ASCC2. These results suggest that ASCC1 coordinates the proper recruitment of the ASCC complex during alkylation, a function that appears to depend on a putative RNA-binding motif near the ASCC1 C terminus. Consistent with its role in alkylation damage signaling and repair, ASCC1 knockout through a CRISPR/Cas9 approach results in alkylation damage sensitivity in a manner epistatic with ASCC3. Together, our results identify a critical regulator of the ALKBH3-ASCC alkylation damage signaling pathway and suggest a potential role for RNA-interacting domains in the alkylation damage response.


Assuntos
DNA Helicases/metabolismo , Proteínas Nucleares/metabolismo , Mapas de Interação de Proteínas , Fatores de Transcrição/metabolismo , Alquilação , Sequência de Aminoácidos , Linhagem Celular , Dano ao DNA , Desmetilação do DNA , Reparo do DNA , Humanos , Modelos Moleculares , Domínios Proteicos , RNA/metabolismo , RNA Ligase (ATP)/química , RNA Ligase (ATP)/metabolismo , Fatores de Transcrição/química
9.
EMBO J ; 34(12): 1687-703, 2015 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-25944111

RESUMO

Repair of DNA alkylation damage is critical for genomic stability and involves multiple conserved enzymatic pathways. Alkylation damage resistance, which is critical in cancer chemotherapy, depends on the overexpression of alkylation repair proteins. However, the mechanisms responsible for this upregulation are unknown. Here, we show that an OTU domain deubiquitinase, OTUD4, is a positive regulator of ALKBH2 and ALKBH3, two DNA demethylases critical for alkylation repair. Remarkably, we find that OTUD4 catalytic activity is completely dispensable for this function. Rather, OTUD4 is a scaffold for USP7 and USP9X, two deubiquitinases that act directly on the AlkB proteins. Moreover, we show that loss of OTUD4, USP7, or USP9X in tumor cells makes them significantly more sensitive to alkylating agents. Taken together, this work reveals a novel, noncanonical mechanism by which an OTU family deubiquitinase regulates its substrates, and provides multiple new targets for alkylation chemotherapy sensitization of tumors.


Assuntos
Alquilação/fisiologia , Dano ao DNA/fisiologia , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA/fisiologia , Dioxigenases/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteases Específicas de Ubiquitina/metabolismo , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato , Alquilação/genética , Western Blotting , Dano ao DNA/genética , Reparo do DNA/genética , Células HEK293 , Humanos , Imunoprecipitação , Microscopia de Fluorescência , Modelos Biológicos , Espectrometria de Massas em Tandem
10.
J Bacteriol ; 198(8): 1268-80, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26833419

RESUMO

UNLABELLED: NADH:quinone oxidoreductase (complex I) is a bioenergetic enzyme that transfers electrons from NADH to quinone, conserving the energy of this reaction by contributing to the proton motive force. While the importance of NADH oxidation to mitochondrial aerobic respiration is well documented, the contribution of complex I to bacterial electron transport chains has been tested in only a few species. Here, we analyze the function of two phylogenetically distinct complex I isozymes in Rhodobacter sphaeroides, an alphaproteobacterium that contains well-characterized electron transport chains. We found that R. sphaeroides complex I activity is important for aerobic respiration and required for anaerobic dimethyl sulfoxide (DMSO) respiration (in the absence of light), photoautotrophic growth, and photoheterotrophic growth (in the absence of an external electron acceptor). Our data also provide insight into the functions of the phylogenetically distinct R. sphaeroidescomplex I enzymes (complex IA and complex IE) in maintaining a cellular redox state during photoheterotrophic growth. We propose that the function of each isozyme during photoheterotrophic growth is either NADH synthesis (complex IA) or NADH oxidation (complex IE). The canonical alphaproteobacterial complex I isozyme (complex IA) was also shown to be important for routing electrons to nitrogenase-mediated H2 production, while the horizontally acquired enzyme (complex IE) was dispensable in this process. Unlike the singular role of complex I in mitochondria, we predict that the phylogenetically distinct complex I enzymes found across bacterial species have evolved to enhance the functions of their respective electron transport chains. IMPORTANCE: Cells use a proton motive force (PMF), NADH, and ATP to support numerous processes. In mitochondria, complex I uses NADH oxidation to generate a PMF, which can drive ATP synthesis. This study analyzed the function of complex I in bacteria, which contain more-diverse and more-flexible electron transport chains than mitochondria. We tested complex I function in Rhodobacter sphaeroides, a bacterium predicted to encode two phylogenetically distinct complex I isozymes. R. sphaeroides cells lacking both isozymes had growth defects during all tested modes of growth, illustrating the important function of this enzyme under diverse conditions. We conclude that the two isozymes are not functionally redundant and predict that phylogenetically distinct complex I enzymes have evolved to support the diverse lifestyles of bacteria.


Assuntos
Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Quinona Redutases/metabolismo , Rhodobacter sphaeroides/enzimologia , Anaerobiose , Hidrogênio/metabolismo , Quinona Redutases/genética , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/metabolismo
11.
bioRxiv ; 2024 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-38559256

RESUMO

Certain environmental toxins are nucleic acid damaging agents, as are many chemotherapeutics used for cancer therapy. These agents induce various adducts in DNA as well as RNA. Indeed, most of the nucleic acid adducts (>90%) formed due to these chemicals, such as alkylating agents, occur in RNA 1 . However, compared to the well-studied mechanisms for DNA alkylation repair, the biological consequences of RNA damage are largely unexplored. Here, we demonstrate that RNA damage can directly result in loss of genome integrity. Specifically, we show that a human YTH domain-containing protein, YTHDC1, regulates alkylation damage responses in association with the THO complex (THOC) 2 . In addition to its established binding to N 6-methyladenosine (m6A)-containing RNAs, YTHDC1 binds to N 1-methyladenosine (m1A)-containing RNAs upon alkylation. In the absence of YTHDC1, alkylation damage results in increased alkylation damage sensitivity and DNA breaks. Such phenotypes are fully attributable to RNA damage, since an RNA-specific dealkylase can rescue these phenotypes. These R NA d amage-induced DNA b reaks (RDIBs) depend on R-loop formation, which in turn are processed by factors involved in transcription-coupled nucleotide excision repair. Strikingly, in the absence of YTHDC1 or THOC, an RNA m1A methyltransferase targeted to the nucleus is sufficient to induce DNA breaks. Our results uncover a unique role for YTHDC1-THOC in base damage responses by preventing RDIBs, providing definitive evidence for how damaged RNAs can impact genomic integrity.

12.
Cancer Discov ; 12(9): 2158-2179, 2022 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-35819319

RESUMO

Small cell lung cancer (SCLC) is the most fatal form of lung cancer, with dismal survival, limited therapeutic options, and rapid development of chemoresistance. We identified the lysine methyltransferase SMYD3 as a major regulator of SCLC sensitivity to alkylation-based chemotherapy. RNF113A methylation by SMYD3 impairs its interaction with the phosphatase PP4, controlling its phosphorylation levels. This cross-talk between posttranslational modifications acts as a key switch in promoting and maintaining RNF113A E3 ligase activity, essential for its role in alkylation damage response. In turn, SMYD3 inhibition restores SCLC vulnerability to alkylating chemotherapy. Our study sheds light on a novel role of SMYD3 in cancer, uncovering this enzyme as a mediator of alkylation damage sensitivity and providing a rationale for small-molecule SMYD3 inhibition to improve responses to established chemotherapy. SIGNIFICANCE: SCLC rapidly becomes resistant to conventional chemotherapy, leaving patients with no alternative treatment options. Our data demonstrate that SMYD3 upregulation and RNF113A methylation in SCLC are key mechanisms that control the alkylation damage response. Notably, SMYD3 inhibition sensitizes cells to alkylating agents and promotes sustained SCLC response to chemotherapy. This article is highlighted in the In This Issue feature, p. 2007.


Assuntos
Proteínas de Ligação a DNA , Histona-Lisina N-Metiltransferase , Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Alquilação , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Metilação , Fosforilação , Processamento de Proteína Pós-Traducional , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/genética
13.
J Cell Biol ; 220(9)2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34232287

RESUMO

R-loops are three-stranded nucleic acid structures with both physiological and pathological roles in cells. R-loop imaging generally relies on detection of the RNA-DNA hybrid component of these structures using the S9.6 antibody. We show that the use of this antibody for imaging can be problematic because it readily binds to double-stranded RNA (dsRNA) in vitro and in vivo, giving rise to nonspecific signal. In contrast, purified, catalytically inactive human RNase H1 tagged with GFP (GFP-dRNH1) is a more specific reagent for imaging RNA-DNA hybrids. GFP-dRNH1 binds strongly to RNA-DNA hybrids but not to dsRNA oligonucleotides in fixed human cells and is not susceptible to binding endogenous RNA. Furthermore, we demonstrate that purified GFP-dRNH1 can be applied to fixed cells to detect hybrids after their induction, thereby bypassing the need for cell line engineering. GFP-dRNH1 therefore promises to be a versatile tool for imaging and quantifying RNA-DNA hybrids under a wide range of conditions.


Assuntos
DNA/metabolismo , Sequências Repetidas Invertidas , RNA de Cadeia Dupla/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Ribonuclease H/metabolismo , Coloração e Rotulagem/métodos , Anticorpos/química , Anticorpos/metabolismo , Proteína BRCA1/antagonistas & inibidores , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Clonagem Molecular , DNA/química , DNA/ultraestrutura , DNA Helicases/antagonistas & inibidores , DNA Helicases/genética , DNA Helicases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/metabolismo , Humanos , Enzimas Multifuncionais/antagonistas & inibidores , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Hibridização de Ácido Nucleico , Imagem Óptica/métodos , Ligação Proteica , RNA Helicases/antagonistas & inibidores , RNA Helicases/genética , RNA Helicases/metabolismo , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/ultraestrutura , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Ribonuclease H/genética
14.
DNA Repair (Amst) ; 81: 102663, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31326362

RESUMO

The response to DNA damage intersects with many other physiological processes in the cell, such as DNA replication, chromatin remodeling, and the cell cycle. Certain damaging lesions, such as UV-induced pyrimidine dimers, also strongly block RNA polymerases, necessitating the coordination of the repair mechanism with remodeling of the elongating transcriptional machinery, in a process called transcription-coupled nucleotide excision repair (TC-NER). This pathway is typically not thought to be engaged with smaller lesions such as base alkylation. However, recent work has uncovered the potential for shared molecular components between the cellular response to alkylation and UV damage. Here, we review our current understanding of the alkylation damage response and its impacts on RNA biogenesis. We give particular attention to the Activating Signal Cointegrator Complex (ASCC), which plays important roles in the transcriptional response during UV damage as well as alkylation damage reversal, and intersects with trichothiodystrophy, an inherited disease associated with TC-NER.


Assuntos
Adutos de DNA/metabolismo , Reparo do DNA , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , Alquilação , Animais , DNA/química , DNA/metabolismo , DNA Helicases/metabolismo , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Transcrição Gênica , Proteínas Supressoras de Tumor/metabolismo
15.
Science ; 362(6415): 694-699, 2018 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-30409884

RESUMO

During the process of cross-presentation, viral or tumor-derived antigens are presented to CD8+ T cells by Batf3-dependent CD8α+/XCR1+ classical dendritic cells (cDC1s). We designed a functional CRISPR screen for previously unknown regulators of cross-presentation, and identified the BEACH domain-containing protein WDFY4 as essential for cross-presentation of cell-associated antigens by cDC1s in mice. However, WDFY4 was not required for major histocompatibility complex class II presentation, nor for cross-presentation by monocyte-derived dendritic cells. In contrast to Batf3 -/- mice, Wdfy4 -/- mice displayed normal lymphoid and nonlymphoid cDC1 populations that produce interleukin-12 and protect against Toxoplasma gondii infection. However, similar to Batf3 -/- mice, Wdfy4 -/- mice failed to prime virus-specific CD8+ T cells in vivo or induce tumor rejection, revealing a critical role for cross-presentation in antiviral and antitumor immunity.


Assuntos
Antígenos de Neoplasias/imunologia , Antígenos Virais/imunologia , Apresentação Cruzada/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/fisiologia , Linfócitos T CD8-Positivos/imunologia , Sistemas CRISPR-Cas , Testes Genéticos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Toxoplasma/imunologia , Toxoplasmose/imunologia
16.
Nat Commun ; 8(1): 860, 2017 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-29038425

RESUMO

The breast cancer susceptibility proteins BRCA1 and BRCA2 have emerged as key stabilizing factors for the maintenance of replication fork integrity following replication stress. In their absence, stalled replication forks are extensively degraded by the MRE11 nuclease, leading to chemotherapeutic sensitivity. Here we report that BRCA proteins prevent nucleolytic degradation by protecting replication forks that have undergone fork reversal upon drug treatment. The unprotected regressed arms of reversed forks are the entry point for MRE11 in BRCA-deficient cells. The CtIP protein initiates MRE11-dependent degradation, which is extended by the EXO1 nuclease. Next, we show that the initial limited resection of the regressed arms establishes the substrate for MUS81 in BRCA2-deficient cells. In turn, MUS81 cleavage of regressed forks with a ssDNA tail promotes POLD3-dependent fork rescue. We propose that targeting this pathway may represent a new strategy to modulate BRCA2-deficient cancer cell response to chemotherapeutics that cause fork degradation.BRCA proteins have emerged as key stabilizing factors for the maintenance of replication forks following replication stress. Here the authors describe how reversed replication forks are degraded in the absence of BRCA2, and a MUS81 and POLD3-dependent mechanism of rescue following the withdrawal of genotoxic agent.


Assuntos
Proteína BRCA2/metabolismo , Proteínas de Transporte/metabolismo , DNA Polimerase III/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Proteína Homóloga a MRE11/metabolismo , Proteínas Nucleares/metabolismo , Linhagem Celular Tumoral , Endodesoxirribonucleases , Recombinação Homóloga , Humanos
17.
Trends Cell Biol ; 24(7): 426-34, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24569222

RESUMO

The cellular response to DNA double-stranded breaks (DSBs) involves a conserved mechanism of recruitment and activation of numerous proteins involved in this pathway. The events that trigger this response in mammalian cells involve several post-translational modifications, but the role of non-proteasomal ubiquitin signaling is particularly central to this pathway. Recent work has demonstrated that ubiquitination does not act alone, but in concert with other post-translational modifications, including phosphorylation, methylation, acetylation, ADP-ribosylation, and other ubiquitin-like modifiers, particularly SUMOylation. We review novel and exciting crosstalk mechanisms between ubiquitination and other post-translational modifications, many of which work synergistically with each other to activate signaling events and help recruit important DNA damage effector proteins, particularly BRCA1 (breast cancer 1, early onset) and 53BP1 (tumor protein p53 binding protein 1), to sites of DNA damage.


Assuntos
Cromatina/genética , Cromatina/metabolismo , Reparo do DNA/genética , Processamento de Proteína Pós-Traducional/genética , Ubiquitina/genética , Ubiquitina/metabolismo , Animais , Quebras de DNA de Cadeia Dupla , Humanos , Transdução de Sinais/genética
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