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1.
EMBO Rep ; : e53543, 2021 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-34842321

RESUMO

Single-cell RNA sequencing (scRNA-seq) is a powerful technique for dissecting the complexity of normal and diseased tissues, enabling characterization of cell diversity and heterogeneous phenotypic states in unprecedented detail. However, this technology has been underutilized for exploring the interactions between the host cell and viral pathogens in latently infected cells. Herein, we use scRNA-seq and single-molecule sensitivity fluorescent in situ hybridization (smFISH) technologies to investigate host single-cell transcriptome changes upon the reactivation of a human neurotropic virus, herpes simplex virus-1 (HSV-1). We identify the stress sensor growth arrest and DNA damage-inducible 45 beta (Gadd45b) as a critical antiviral host factor that regulates HSV-1 reactivation events in a subpopulation of latently infected primary neurons. We show that distinct subcellular localization of Gadd45b correlates with the viral late gene expression program, as well as the expression of the viral transcription factor, ICP4. We propose that a hallmark of a "successful" or "aborted" HSV-1 reactivation state in primary neurons is determined by a unique subcellular localization signature of the stress sensor Gadd45b.

2.
Cell Rep Med ; 2(9): 100402, 2021 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-34622238

RESUMO

CCNE1 amplification is an oncogenic driver for many gynecologic cancers and is associated with poor patient outcomes. In this issue, Xu et al.1 identify a combination therapy that is responsive to high CCNE1-copy number ovarian and endometrial cancers using PDX models.

3.
Cell Rep ; 36(13): 109754, 2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34547223

RESUMO

The SARS-CoV-2 papain-like protease (PLpro) is a target for antiviral drug development. It is essential for processing viral polyproteins for replication and functions in host immune evasion by cleaving ubiquitin (Ub) and ubiquitin-like protein (Ubl) conjugates. While highly conserved, SARS-CoV-2 and SARS-CoV PLpro have contrasting Ub/Ubl substrate preferences. Using a combination of structural analyses and functional assays, we identify a molecular sensor within the S1 Ub-binding site of PLpro that serves as a key determinant of substrate specificity. Variations within the S1 sensor specifically alter cleavage of Ub substrates but not of the Ubl interferon-stimulated gene 15 protein (ISG15). Significantly, a variant of concern associated with immune evasion carries a mutation in the S1 sensor that enhances PLpro activity on Ub substrates. Collectively, our data identify the S1 sensor region as a potential hotspot of variability that could alter host antiviral immune responses to newly emerging SARS-CoV-2 lineages.


Assuntos
Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Proteases Semelhantes à Papaína de Coronavírus/ultraestrutura , SARS-CoV-2/genética , Sequência de Aminoácidos/genética , Sítios de Ligação/genética , COVID-19/genética , COVID-19/metabolismo , Proteases Semelhantes à Papaína de Coronavírus/genética , Células HEK293 , Humanos , Papaína/química , Papaína/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Ligação Proteica/genética , SARS-CoV-2/metabolismo , Especificidade por Substrato/genética , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Proteínas Virais/metabolismo
4.
Mol Cell ; 81(20): 4243-4257.e6, 2021 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-34473946

RESUMO

Mammalian cells use diverse pathways to prevent deleterious consequences during DNA replication, yet the mechanism by which cells survey individual replisomes to detect spontaneous replication impediments at the basal level, and their accumulation during replication stress, remain undefined. Here, we used single-molecule localization microscopy coupled with high-order-correlation image-mining algorithms to quantify the composition of individual replisomes in single cells during unperturbed replication and under replicative stress. We identified a basal-level activity of ATR that monitors and regulates the amounts of RPA at forks during normal replication. Replication-stress amplifies the basal activity through the increased volume of ATR-RPA interaction and diffusion-driven enrichment of ATR at forks. This localized crowding of ATR enhances its collision probability, stimulating the activation of its replication-stress response. Finally, we provide a computational model describing how the basal activity of ATR is amplified to produce its canonical replication stress response.

5.
J Biol Chem ; 297(3): 101049, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34375640

RESUMO

Fused in sarcoma (FUS) encodes an RNA-binding protein with diverse roles in transcriptional activation and RNA splicing. While oncogenic fusions of FUS and transcription factor DNA-binding domains are associated with soft tissue sarcomas, dominant mutations in FUS can cause amyotrophic lateral sclerosis. FUS has also been implicated in genome maintenance. However, the underlying mechanisms of its actions in genome stability are unknown. Here, we applied gene editing, functional reconstitution, and integrated proteomics and transcriptomics to illuminate roles for FUS in DNA replication and repair. Consistent with a supportive role in DNA double-strand break repair, FUS-deficient cells exhibited subtle alterations in the recruitment and retention of double-strand break-associated factors, including 53BP1 and BRCA1. FUS-/- cells also exhibited reduced proliferative potential that correlated with reduced speed of replication fork progression, diminished loading of prereplication complexes, enhanced micronucleus formation, and attenuated expression and splicing of S-phase-associated genes. Finally, FUS-deficient cells exhibited genome-wide alterations in DNA replication timing that were reversed upon re-expression of FUS complementary DNA. We also showed that FUS-dependent replication domains were enriched in transcriptionally active chromatin and that FUS was required for the timely replication of transcriptionally active DNA. These findings suggest that alterations in DNA replication kinetics and programming contribute to genome instability and functional defects in FUS-deficient cells.

6.
Dev Cell ; 56(15): 2207-2222.e7, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34256011

RESUMO

Cells counter DNA damage through repair or apoptosis, yet a direct mechanism for this choice has remained elusive. When facing interstrand crosslinks (ICLs), the ICL-repair protein FANCI heterodimerizes with FANCD2 to initiate ICL excision. We found that FANCI alternatively interacts with a pro-apoptotic factor, PIDD1, to enable PIDDosome (PIDD1-RAIDD-caspase-2) formation and apoptotic death. FANCI switches from FANCD2/repair to PIDD1/apoptosis signaling in the event of ICL-repair failure. Specifically, removing key endonucleases downstream of FANCI/FANCD2, increasing ICL levels, or allowing damaged cells into mitosis (when repair is suppressed) all suffice for switching. Reciprocally, apoptosis-committed FANCI reverts from PIDD1 to FANCD2 after a failed attempt to assemble the PIDDosome. Monoubiquitination and deubiquitination at FANCI K523 impact interactor selection. These data unveil a repair-or-apoptosis switch in eukaryotes. Beyond ensuring the removal of unrepaired genomes, the switch's bidirectionality reveals that damaged cells can offset apoptotic defects via de novo attempts at lesion repair.


Assuntos
Apoptose/fisiologia , Reparo do DNA/fisiologia , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Animais , Proteína Adaptadora de Sinalização CRADD/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , DNA/metabolismo , Dano ao DNA/fisiologia , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/fisiologia , Proteínas de Grupos de Complementação da Anemia de Fanconi/fisiologia , Células HeLa , Humanos , Ubiquitinação , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
7.
Nat Commun ; 12(1): 2525, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33953191

RESUMO

Guanine-rich DNA sequences occur throughout the human genome and can transiently form G-quadruplex (G4) structures that may obstruct DNA replication, leading to genomic instability. Here, we apply multi-color single-molecule localization microscopy (SMLM) coupled with robust data-mining algorithms to quantitatively visualize replication fork (RF)-coupled formation and spatial-association of endogenous G4s. Using this data, we investigate the effects of G4s on replisome dynamics and organization. We show that a small fraction of active replication forks spontaneously form G4s at newly unwound DNA immediately behind the MCM helicase and before nascent DNA synthesis. These G4s locally perturb replisome dynamics and organization by reducing DNA synthesis and limiting the binding of the single-strand DNA-binding protein RPA. We find that the resolution of RF-coupled G4s is mediated by an interplay between RPA and the FANCJ helicase. FANCJ deficiency leads to G4 accumulation, DNA damage at G4-associated replication forks, and silencing of the RPA-mediated replication stress response. Our study provides first-hand evidence of the intrinsic, RF-coupled formation of G4 structures, offering unique mechanistic insights into the interference and regulation of stable G4s at replication forks and their effect on RPA-associated fork signaling and genomic instability.


Assuntos
Replicação do DNA/fisiologia , DNA/química , Quadruplex G , Imagem Individual de Molécula/métodos , Animais , Biofísica , Linhagem Celular , Dano ao DNA , DNA Helicases/metabolismo , Proteínas de Ligação a DNA , Instabilidade Genômica , Humanos , Proteínas Recombinantes , Células Sf9
8.
Nat Protoc ; 16(2): 1193-1218, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33442052

RESUMO

The ability to monitor DNA replication fork directionality at the genome-wide scale is paramount for a greater understanding of how genetic and environmental perturbations can impact replication dynamics in human cells. Here we describe a detailed protocol for isolating and sequencing Okazaki fragments from asynchronously growing mammalian cells, termed Okazaki fragment sequencing (Ok-seq), for the purpose of quantitatively determining replication initiation and termination frequencies around specific genomic loci by meta-analyses. Briefly, cells are pulsed with 5-ethynyl-2'-deoxyuridine (EdU) to label newly synthesized DNA, and collected for DNA extraction. After size fractionation on a sucrose gradient, Okazaki fragments are concentrated and purified before click chemistry is used to tag the EdU label with a biotin conjugate that is cleavable under mild conditions. Biotinylated Okazaki fragments are then captured on streptavidin beads and ligated to Illumina adapters before library preparation for Illumina sequencing. The use of Ok-seq to interrogate genome-wide replication fork initiation and termination efficiencies can be applied to all unperturbed, asynchronously growing mammalian cells or under conditions of replication stress, and the assay can be performed in less than 2 weeks.


Assuntos
Replicação do DNA/fisiologia , DNA/análise , Química Click/métodos , DNA/genética , Replicação do DNA/genética , Desoxiuridina/análogos & derivados , Desoxiuridina/química , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Estreptavidina
9.
Mol Cell ; 80(4): 682-698.e7, 2020 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-33152268

RESUMO

Knowledge of fundamental differences between breast cancer subtypes has driven therapeutic advances; however, basal-like breast cancer (BLBC) remains clinically intractable. Because BLBC exhibits alterations in DNA repair enzymes and cell-cycle checkpoints, elucidation of factors enabling the genomic instability present in this subtype has the potential to reveal novel anti-cancer strategies. Here, we demonstrate that BLBC is especially sensitive to suppression of iron-sulfur cluster (ISC) biosynthesis and identify DNA polymerase epsilon (POLE) as an ISC-containing protein that underlies this phenotype. In BLBC cells, POLE suppression leads to replication fork stalling, DNA damage, and a senescence-like state or cell death. In contrast, luminal breast cancer and non-transformed mammary cells maintain viability upon POLE suppression but become dependent upon an ATR/CHK1/CDC25A/CDK2 DNA damage response axis. We find that CDK1/2 targets exhibit hyperphosphorylation selectively in BLBC tumors, indicating that CDK2 hyperactivity is a genome integrity vulnerability exploitable by targeting POLE.


Assuntos
Neoplasias da Mama/patologia , Carcinoma Basocelular/patologia , Quinase 2 Dependente de Ciclina/metabolismo , DNA Polimerase II/metabolismo , Instabilidade Genômica , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Animais , Apoptose , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinoma Basocelular/genética , Carcinoma Basocelular/metabolismo , Ciclo Celular , Proliferação de Células , Quinase 2 Dependente de Ciclina/genética , Dano ao DNA , DNA Polimerase II/genética , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose/genética , Transdução de Sinais , Células Tumorais Cultivadas
10.
Sci Adv ; 6(42)2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33067239

RESUMO

Viral papain-like cysteine protease (PLpro, NSP3) is essential for SARS-CoV-2 replication and represents a promising target for the development of antiviral drugs. Here, we used a combinatorial substrate library and performed comprehensive activity profiling of SARS-CoV-2 PLpro. On the scaffold of the best hits from positional scanning, we designed optimal fluorogenic substrates and irreversible inhibitors with a high degree of selectivity for SARS PLpro. We determined crystal structures of two of these inhibitors in complex with SARS-CoV-2 PLpro that reveals their inhibitory mechanisms and provides a molecular basis for the observed substrate specificity profiles. Last, we demonstrate that SARS-CoV-2 PLpro harbors deISGylating activity similar to SARSCoV-1 PLpro but its ability to hydrolyze K48-linked Ub chains is diminished, which our sequence and structure analysis provides a basis for. Together, this work has revealed the molecular rules governing PLpro substrate specificity and provides a framework for development of inhibitors with potential therapeutic value or drug repurposing.


Assuntos
Betacoronavirus/enzimologia , Desenho de Fármacos , Inibidores de Proteases/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Sequência de Aminoácidos , Betacoronavirus/isolamento & purificação , Sítios de Ligação , COVID-19 , Domínio Catalítico , Proteases 3C de Coronavírus , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Cristalografia por Raios X , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Humanos , Cinética , Simulação de Dinâmica Molecular , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Inibidores de Proteases/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , SARS-CoV-2 , Especificidade por Substrato , Ubiquitinas/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
11.
DNA Repair (Amst) ; 95: 102948, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32866776

RESUMO

The DNA damage response (DDR) is necessary to maintain genome integrity and prevent the accumulation of oncogenic mutations. Consequently, proteins involved in the DDR often serve as tumor suppressors, carrying out the crucial task of keeping DNA fidelity intact. Mediator of DNA damage checkpoint 1 (MDC1) is a scaffold protein involved in the early steps of the DDR. MDC1 interacts directly with γ-H2AX, the phosphorylated form of H2AX, a commonly used marker for DNA damage. It then propagates the phosphorylation of H2AX by recruiting ATM kinase. While the function of MDC1 in the DDR has been reviewed previously, its role in cancer has not been reviewed, and numerous studies have recently identified a link between MDC1 and carcinogenesis. This includes MDC1 functioning as a tumor suppressor, with its loss serving as a biomarker for cancer and contributor to drug sensitivity. Studies also indicate that MDC1 operates outside of its traditional role in DDR, and functions as a co-regulator of nuclear receptor transcriptional activity, and that mutations in MDC1 are present in tumors and can also cause germline predisposition to cancer. This review will discuss reports that link MDC1 to cancer and identify MDC1 as an important player in tumor formation, progression, and treatment. We also discuss mechanisms by which MDC1 levels are regulated and how this contributes to tumor formation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinogênese , Proteínas de Ciclo Celular/metabolismo , Neoplasias/metabolismo , Neoplasias/terapia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular/genética , Humanos , Mutação , Neoplasias/genética , Neoplasias/patologia , Transcrição Genética
12.
Chem Sci ; 11(23): 6058-6069, 2020 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-32953009

RESUMO

Deubiquitinating enzymes (DUBs) are responsible for removing ubiquitin (Ub) from its protein conjugates. DUBs have been implicated as attractive therapeutic targets in the treatment of viral diseases, neurodegenerative disorders and cancer. The lack of selective chemical tools for the exploration of these enzymes significantly impairs the determination of their roles in both normal and pathological states. Commercially available fluorogenic substrates are based on the C-terminal Ub motif or contain Ub coupled to a fluorophore (Z-LRGG-AMC, Ub-AMC); therefore, these substrates suffer from lack of selectivity. By using a hybrid combinatorial substrate library (HyCoSuL) and a defined P2 library containing a wide variety of nonproteinogenic amino acids, we established a full substrate specificity profile for two DUBs-MERS PLpro and human UCH-L3. Based on these results, we designed and synthesized Ub-based substrates and activity-based probes (ABPs) containing selected unnatural amino acids located in the C-terminal Ub motif. Biochemical analysis and cell lysate experiments confirmed the activity and selectivity of engineered Ub-based substrates and probes. Using this approach, we propose that for any protease that recognizes Ub and Ub-like substrates, a highly active and selective unnatural substrate or probe can be engineered.

13.
bioRxiv ; 2020 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-32511411

RESUMO

In December 2019, the first cases of a novel coronavirus infection causing COVID-19 were diagnosed in Wuhan, China. Viral Papain-Like cysteine protease (PLpro, NSP3) is essential for SARS-CoV-2 replication and represents a promising target for the development of antiviral drugs. Here, we used a combinatorial substrate library containing natural and a wide variety of nonproteinogenic amino acids and performed comprehensive activity profiling of SARS-CoV-2-PLpro. On the scaffold of best hits from positional scanning we designed optimal fluorogenic substrates and irreversible inhibitors with a high degree of selectivity for SARS PLpro variants versus other proteases. We determined crystal structures of two of these inhibitors (VIR250 and VIR251) in complex with SARS-CoV-2-PLpro which reveals their inhibitory mechanisms and provides a structural basis for the observed substrate specificity profiles. Lastly, we demonstrate that SARS-CoV-2-PLpro harbors deISGylating activities similar to SARS-CoV-1-PLpro but its ability to hydrolyze K48-linked Ub chains is diminished, which our sequence and structure analysis provides a basis for. Altogether this work has revealed the molecular rules governing PLpro substrate specificity and provides a framework for development of inhibitors with potential therapeutic value or drug repositioning.

14.
J Plast Reconstr Aesthet Surg ; 73(10): 1815-1824, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32593571

RESUMO

BACKGROUND: The management of chronic pulmonary aspergillosis remains a challenge for thoracic and reconstructive surgeons. Different management options have been proposed with no consensus regarding the best treatment modality. The goal of this study is to report our experience with the use of intrathoracic muscle flaps for the management of pulmonary aspergillosis. METHODS: We retrospectively reviewed all patients who underwent intrathoracic muscle flap transposition for the management of pulmonary aspergillosis between 1990 and 2010. Demographics, surgical characteristics, and treatment outcomes were collected and analyzed. RESULTS: A total of 39 patients who underwent 48 muscle flaps were identified. The majority were classified as ASA 3 (n=30, 77%) or ASA 4 (n=8, 21%). Serratus anterior was the most common flap used (n=34), followed by latissimus dorsi (n=6) and pectoralis major (n=5). Flap loss was encountered in three (8%) patients (2 partial, 1 total). Bronchopleural fistula and empyema comprised the two most common intrathoracic complications (26%, 29% respectively). Median follow-up was 33 months (range, 0-216). Successful treatment was achieved in 77% of patients, while operative mortality was 23%. CONCLUSION: The use of intrathoracic muscle flaps can be a helpful adjunct to surgical resection in the treatment of chronic pulmonary aspergillosis with low rates of flap loss.


Assuntos
Aspergilose Pulmonar/cirurgia , Procedimentos Cirúrgicos Reconstrutivos/métodos , Retalhos Cirúrgicos , Idoso , Doença Crônica , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/transplante , Complicações Pós-Operatórias/epidemiologia , Estudos Retrospectivos , Parede Torácica
15.
PLoS Genet ; 16(3): e1008524, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32142505

RESUMO

Common fragile sites (CFSs) are breakage-prone genomic loci, and are considered to be hotspots for genomic rearrangements frequently observed in cancers. Understanding the underlying mechanisms for CFS instability will lead to better insight on cancer etiology. Here we show that Polycomb group proteins BMI1 and RNF2 are suppressors of transcription-replication conflicts (TRCs) and CFS instability. Cells depleted of BMI1 or RNF2 showed slower replication forks and elevated fork stalling. These phenotypes are associated with increase occupancy of RNA Pol II (RNAPII) at CFSs, suggesting that the BMI1-RNF2 complex regulate RNAPII elongation at these fragile regions. Using proximity ligase assays, we showed that depleting BMI1 or RNF2 causes increased associations between RNAPII with EdU-labeled nascent forks and replisomes, suggesting increased TRC incidences. Increased occupancy of a fork protective factor FANCD2 and R-loop resolvase RNH1 at CFSs are observed in RNF2 CRISPR-KO cells, which are consistent with increased transcription-associated replication stress in RNF2-deficient cells. Depleting FANCD2 or FANCI proteins further increased genomic instability and cell death of the RNF2-deficient cells, suggesting that in the absence of RNF2, cells depend on these fork-protective factors for survival. These data suggest that the Polycomb proteins have non-canonical roles in suppressing TRC and preserving genomic integrity.


Assuntos
Sítios Frágeis do Cromossomo/genética , Replicação do DNA/genética , Complexo Repressor Polycomb 1/genética , Transcrição Genética/genética , Linhagem Celular , Linhagem Celular Tumoral , Instabilidade Genômica/genética , Células HEK293 , Células HeLa , Humanos
16.
Methods Mol Biol ; 2060: 263-277, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31617183

RESUMO

We describe a primary neuronal culture system suitable for molecular characterization of herpes simplex virus type 1 (HSV-1) infection, latency, and reactivation. While several alternative models are available, including infections of live animal or explanted ganglia, these are complicated by the presence of multiple cell types, including immune cells, and difficulties in manipulating the neuronal environment. The highly pure neuron culture system described here can be readily manipulated and is ideal for molecular studies that focus exclusively on the relationship between the virus and host neuron, the fundamental unit of latency. As such this model allows for detailed investigations of both viral and neuronal factors involved in the establishment and maintenance of HSV-1 latency and in viral reactivation induced by defined stimuli.


Assuntos
Técnicas de Cultura de Células , Herpesvirus Humano 1/fisiologia , Neurônios , Ativação Viral/fisiologia , Latência Viral/fisiologia , Animais , Células Cultivadas , Neurônios/metabolismo , Neurônios/patologia , Neurônios/virologia , Ratos , Ratos Sprague-Dawley
17.
DNA Repair (Amst) ; 78: 20-26, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30954011

RESUMO

DNA replication stress, defined as the slowing or stalling of replication forks, is considered an emerging hallmark of cancer and a major contributor to genomic instability associated with tumorigenesis (Macheret and Halazonetis, 2015). Recent advances have been made in attempting to target DNA repair factors involved in alleviating replication stress to potentiate genotoxic treatments. Various inhibitors of ATR and Chk1, the two major kinases involved in the intra-S-phase checkpoint, are currently in Phase I and II clinical trials [2]. In addition, currently approved inhibitors of Poly-ADP Ribose Polymerase (PARP) show synthetic lethality in cells that lack double-strand break repair such as in BRCA1/2 deficient tumors [3]. These drugs have also been shown to exacerbate replication stress by creating a DNA-protein crosslink, termed PARP 'trapping', and this is now thought to contribute to the therapeutic efficacy. Translesion synthesis (TLS) is a mechanism whereby special repair DNA polymerases accommodate and tolerate various DNA lesions to allow for damage bypass and continuation of DNA replication (Yang and Gao, 2018). This class of proteins is best characterized by the Y-family, encompassing DNA polymerases (Pols) Kappa, Eta, Iota, and Rev1. While best studied for their ability to bypass physical lesions on the DNA, there is accumulating evidence for these proteins in coping with various natural replication fork barriers and alleviating replication stress. In this mini-review, we will highlight some of these recent advances, and discuss why targeting the TLS pathway may be a mechanism of enhancing cancer-associated replication stress. Exacerbation of replication stress can lead to increased genome instability, which can be toxic to cancer cells and represent a therapeutic vulnerability.


Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Terapia de Alvo Molecular/métodos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Estresse Fisiológico/genética , Animais , Replicação do DNA/efeitos dos fármacos , Humanos , Neoplasias/enzimologia , Estresse Fisiológico/efeitos dos fármacos
18.
Mol Cell ; 74(3): 466-480.e4, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30930055

RESUMO

The mTOR pathway integrates both extracellular and intracellular signals and serves as a central regulator of cell metabolism, growth, survival, and stress responses. Neurotropic viruses, such as herpes simplex virus-1 (HSV-1), also rely on cellular AKT-mTORC1 signaling to achieve viral latency. Here, we define a novel genotoxic response whereby spatially separated signals initiated by extracellular neurotrophic factors and nuclear DNA damage are integrated by the AKT-mTORC1 pathway. We demonstrate that endogenous DNA double-strand breaks (DSBs) mediated by Topoisomerase 2ß-DNA cleavage complex (TOP2ßcc) intermediates are required to achieve AKT-mTORC1 signaling and maintain HSV-1 latency in neurons. Suppression of host DNA-repair pathways that remove TOP2ßcc trigger HSV-1 reactivation. Moreover, perturbation of AKT phosphorylation dynamics by downregulating the PHLPP1 phosphatase led to AKT mis-localization and disruption of DSB-induced HSV-1 reactivation. Thus, the cellular genome integrity and environmental inputs are consolidated and co-opted by a latent virus to balance lifelong infection with transmission.


Assuntos
DNA Topoisomerases Tipo II/genética , Herpesvirus Humano 1/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-akt/genética , Latência Viral/genética , Animais , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Herpesvirus Humano 1/patogenicidade , Humanos , Proteína Homóloga a MRE11/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Neurônios/metabolismo , Neurônios/virologia , Fosforilação , Ratos , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética
19.
Nat Struct Mol Biol ; 26(1): 67-77, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30598550

RESUMO

Although DNA replication is a fundamental aspect of biology, it is not known what determines where DNA replication starts and stops in the human genome. We directly identified and quantitatively compared sites of replication initiation and termination in untransformed human cells. We found that replication preferentially initiates at the transcription start site of genes occupied by high levels of RNA polymerase II, and terminates at their polyadenylation sites, thereby ensuring global co-directionality of transcription and replication, particularly at gene 5' ends. During replication stress, replication initiation is stimulated downstream of genes and termination is redistributed to gene bodies; this globally reorients replication relative to transcription around gene 3' ends. These data suggest that replication initiation and termination are coupled to transcription in human cells, and propose a model for the impact of replication stress on genome integrity.


Assuntos
Replicação do DNA/genética , Origem de Replicação/genética , Transcrição Genética/genética , Replicação do DNA/fisiologia , Humanos , Poliadenilação/genética , Poliadenilação/fisiologia , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Origem de Replicação/fisiologia , Sítio de Iniciação de Transcrição/fisiologia , Transcrição Genética/fisiologia
20.
Cancer Res ; 79(1): 72-85, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30425057

RESUMO

Aberrant activation of ß-catenin signaling is a critical driver for tumorigenesis, but the mechanism underlying this activation is not completely understood. In this study, we demonstrate a critical role of ß-catenin signaling in stabilization of enhancer of zeste homolog 2 (EZH2) and control of EZH2-mediated gene repression in oncogenesis. ß-Catenin/TCF4 activated the transcription of the deubiquitinase USP1, which then interacted with and deubiquitinated EZH2 directly. USP1-mediated stabilization of EZH2 promoted its recruitment to the promoters of CDKN1B, RUNX3, and HOXA5, resulting in enhanced enrichment of histone H3K27me3 and repression of target gene expression. In human glioma specimens, expression levels of nuclear ß-catenin, USP1, and EZH2 correlated with one another. Depletion of ß-catenin/USP1/EZH2 repressed glioma cell proliferation in vitro and tumor formation in vivo. Our findings indicate that a ß-catenin-USP1-EZH2 axis orchestrates the interplay between dysregulated ß-catenin signaling and EZH2-mediated gene epigenetic silencing during glioma tumorigenesis. SIGNIFICANCE: These findings identify the ß-catenin-USP1-EZH2 signaling axis as a critical mechanism for glioma tumorigenesis that may serve as a new therapeutic target in glioblastoma.


Assuntos
Carcinogênese/patologia , Proteína Potenciadora do Homólogo 2 de Zeste/química , Regulação Neoplásica da Expressão Gênica , Glioma/patologia , Proteases Específicas de Ubiquitina/metabolismo , beta Catenina/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Proliferação de Células , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Epigênese Genética , Glioma/genética , Glioma/metabolismo , Humanos , Camundongos , Camundongos Nus , Prognóstico , Estabilidade Proteica , Taxa de Sobrevida , Células Tumorais Cultivadas , Proteases Específicas de Ubiquitina/genética , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genética
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