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1.
Nat Commun ; 11(1): 4709, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948765

RESUMO

Glioblastoma cancer-stem like cells (GSCs) display marked resistance to ionizing radiation (IR), a standard of care for glioblastoma patients. Mechanisms underpinning radio-resistance of GSCs remain largely unknown. Chromatin state and the accessibility of DNA lesions to DNA repair machineries are crucial for the maintenance of genomic stability. Understanding the functional impact of chromatin remodeling on DNA repair in GSCs may lay the foundation for advancing the efficacy of radio-sensitizing therapies. Here, we present the results of a high-content siRNA microscopy screen, revealing the transcriptional elongation factor SPT6 to be critical for the genomic stability and self-renewal of GSCs. Mechanistically, SPT6 transcriptionally up-regulates BRCA1 and thereby drives an error-free DNA repair in GSCs. SPT6 loss impairs the self-renewal, genomic stability and tumor initiating capacity of GSCs. Collectively, our results provide mechanistic insights into how SPT6 regulates DNA repair and identify SPT6 as a putative therapeutic target in glioblastoma.


Assuntos
Reparo do DNA , Instabilidade Genômica , Glioblastoma/genética , Células-Tronco Neoplásicas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Apoptose , Proteína BRCA1 , Neoplasias Encefálicas/genética , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Glioblastoma/patologia , Células HEK293 , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Células-Tronco Neoplásicas/patologia , RNA Interferente Pequeno/genética , Tolerância a Radiação , Radiação Ionizante , Transcriptoma
2.
Mol Cell ; 79(6): 1037-1050.e5, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32882183

RESUMO

DNA double-stranded breaks (DSBs) are dangerous lesions threatening genomic stability. Fidelity of DSB repair is best achieved by recombination with a homologous template sequence. In yeast, transcript RNA was shown to template DSB repair of DNA. However, molecular pathways of RNA-driven repair processes remain obscure. Utilizing assays of RNA-DNA recombination with and without an induced DSB in yeast DNA, we characterize three forms of RNA-mediated genomic modifications: RNA- and cDNA-templated DSB repair (R-TDR and c-TDR) using an RNA transcript or a DNA copy of the RNA transcript for DSB repair, respectively, and a new mechanism of RNA-templated DNA modification (R-TDM) induced by spontaneous or mutagen-induced breaks. While c-TDR requires reverse transcriptase, translesion DNA polymerase ζ (Pol ζ) plays a major role in R-TDR, and it is essential for R-TDM. This study characterizes mechanisms of RNA-DNA recombination, uncovering a role of Pol ζ in transferring genetic information from transcript RNA to DNA.


Assuntos
DNA/genética , RNA/genética , Saccharomyces cerevisiae/genética , Adolescente , Adulto , DNA/ultraestrutura , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Replicação do DNA/genética , DNA Complementar/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/ultraestrutura , Instabilidade Genômica/genética , Humanos , Pessoa de Meia-Idade , RNA/ultraestrutura , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Adulto Jovem
3.
Nat Commun ; 11(1): 4515, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908127

RESUMO

The discovery of ancestral RAG transposons in early deuterostomia reveals the origin of vertebrate V(D)J recombination. Here, we analyze the functional regulation of a RAG transposon, ProtoRAG, in lancelet. We find that a specific interaction between the cis-acting element within the TIR sequences of ProtoRAG and a trans-acting factor, lancelet YY1-like (bbYY1), is important for the transcriptional regulation of lancelet RAG-like genes (bbRAG1L and bbRAG2L). Mechanistically, bbYY1 suppresses the transposition of ProtoRAG; meanwhile, bbYY1 promotes host DNA rejoins (HDJ) and TIR-TIR joints (TTJ) after TIR-dependent excision by facilitating the binding of bbRAG1L/2 L to TIR-containing DNA, and by interacting with the bbRAG1L/2 L complex. Our data thus suggest that bbYY1 has dual functions in fine-tuning the activity of ProtoRAG and maintaining the genome stability of the host.


Assuntos
Elementos de DNA Transponíveis/genética , Proteínas de Ligação a DNA/metabolismo , Anfioxos/genética , Recombinação V(D)J , Fator de Transcrição YY1/metabolismo , Animais , Técnicas de Silenciamento de Genes , Genes RAG-1 , Instabilidade Genômica , Células HEK293 , Células HeLa , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/isolamento & purificação
4.
Nat Commun ; 11(1): 4534, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913330

RESUMO

Collisions between the DNA replication machinery and co-transcriptional R-loops can impede DNA synthesis and are a major source of genomic instability in cancer cells. How cancer cells deal with R-loops to proliferate is poorly understood. Here we show that the ATP-dependent chromatin remodelling INO80 complex promotes resolution of R-loops to prevent replication-associated DNA damage in cancer cells. Depletion of INO80 in prostate cancer PC3 cells leads to increased R-loops. Overexpression of the RNA:DNA endonuclease RNAse H1 rescues the DNA synthesis defects and suppresses DNA damage caused by INO80 depletion. R-loops co-localize with and promote recruitment of INO80 to chromatin. Artificial tethering of INO80 to a LacO locus enabled turnover of R-loops in cis. Finally, counteracting R-loops by INO80 promotes proliferation and averts DNA damage-induced death in cancer cells. Our work suggests that INO80-dependent resolution of R-loops promotes DNA replication in the presence of transcription, thus enabling unlimited proliferation in cancers.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proliferação de Células/genética , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Neoplasias/genética , Estruturas R-Loop/genética , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Montagem e Desmontagem da Cromatina , Dano ao DNA , Instabilidade Genômica , Humanos , Neoplasias/patologia , Transcrição Genética
5.
Sci Total Environ ; 745: 141048, 2020 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-32758757

RESUMO

Human activity is a serious cause of extensive changes in the environment and a constant reason for the emergence of new stress factors. Thus, to survive and reproduce, organisms must constantly implement a program of adaptation to continuously changing conditions. The research presented here is focused on tracking slow changes occurring in Spodoptera exigua (Lepidoptera: Noctuidae) caused by multigenerational exposure to sub-lethal cadmium doses. The insects received food containing cadmium at concentrations of 5, 11, 22 and 44 µg per g of dry mass of food. The level of DNA stability was monitored by a comet assay in subsequent generations up to the 36th generation. In the first three generations, the level of DNA damage was high, especially in the groups receiving higher doses of cadmium in the diet. In the fourth generation, a significant reduction in the level of DNA damage was observed, which could indicate that the desired stability of the genome was achieved. Surprisingly, however, in subsequent generations, an alternating increase and decrease was found in DNA stability. The observed cycles of changing DNA stability were longer lasting in insects consuming food with a lower Cd content. Thus, a transient reduction in genome stability can be perceived as an opportunity to increase the number of genotypes that undergo selection. This phenomenon occurs faster if the severity of the stress factor is high but is low enough to allow the population to survive.


Assuntos
Cádmio/toxicidade , Dano ao DNA , Animais , Instabilidade Genômica , Humanos , Larva , Spodoptera/genética
6.
PLoS One ; 15(8): e0237559, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32780783

RESUMO

BACKGROUND: The world is going through the critical phase of COVID-19 pandemic, caused by human coronavirus, SARS-CoV-2. Worldwide concerted effort to identify viral genomic changes across different sub-types has identified several strong changes in the coding region. However, there have not been many studies focusing on the variations in the 5' and 3' untranslated regions and their consequences. Considering the possible importance of these regions in host mediated regulation of viral RNA genome, we wanted to explore the phenomenon. METHODS: To have an idea of the global changes in 5' and 3'-UTR sequences, we downloaded 8595 complete and high-coverage SARS-CoV-2 genome sequence information from human host in FASTA format from Global Initiative on Sharing All Influenza Data (GISAID) from 15 different geographical regions. Next, we aligned them using Clustal Omega software and investigated the UTR variants. We also looked at the putative host RNA binding protein (RBP) and microRNA binding sites in these regions by 'RBPmap' and 'RNA22 v2' respectively. Expression status of selected RBPs and microRNAs were checked in lungs tissue. RESULTS: We identified 28 unique variants in SARS-CoV-2 UTR region based on a minimum variant percentage cut-off of 0.5. Along with 241C>T change the important 5'-UTR change identified was 187A>G, while 29734G>C, 29742G>A/T and 29774C>T were the most familiar variants of 3'UTR among most of the continents. Furthermore, we found that despite the variations in the UTR regions, binding of host RBP to them remains mostly unaltered, which further influenced the functioning of specific miRNAs. CONCLUSION: Our results, shows for the first time in SARS-Cov-2 infection, a possible cross-talk between host RBPs-miRNAs and viral UTR variants, which ultimately could explain the mechanism of escaping host RNA decay machinery by the virus. The knowledge might be helpful in developing anti-viral compounds in future.


Assuntos
Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Betacoronavirus/genética , Infecções por Coronavirus/metabolismo , Genoma Viral/genética , Instabilidade Genômica/genética , Interações Hospedeiro-Patógeno/genética , MicroRNAs/metabolismo , Pneumonia Viral/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequência de Bases , Sítios de Ligação , Infecções por Coronavirus/virologia , Humanos , Fases de Leitura Aberta/genética , Pandemias , Pneumonia Viral/virologia , Ligação Proteica/genética
7.
Nat Commun ; 11(1): 4330, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859912

RESUMO

Sex differences have been observed in multiple facets of cancer epidemiology, treatment and biology, and in most cancers outside the sex organs. Efforts to link these clinical differences to specific molecular features have focused on somatic mutations within the coding regions of the genome. Here we report a pan-cancer analysis of sex differences in whole genomes of 1983 tumours of 28 subtypes as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium. We both confirm the results of exome studies, and also uncover previously undescribed sex differences. These include sex-biases in coding and non-coding cancer drivers, mutation prevalence and strikingly, in mutational signatures related to underlying mutational processes. These results underline the pervasiveness of molecular sex differences and strengthen the call for increased consideration of sex in molecular cancer research.


Assuntos
Mutação , Neoplasias/genética , Oncogenes/genética , Caracteres Sexuais , Instabilidade Cromossômica , Exoma , Feminino , Genoma Humano , Instabilidade Genômica , Humanos , Modelos Logísticos , Masculino , Fases de Leitura Aberta , beta Catenina/genética
8.
Nat Commun ; 11(1): 4083, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796829

RESUMO

Proper chromatin function and maintenance of genomic stability depends on spatiotemporal coordination between the transcription and replication machinery. Loss of this coordination can lead to DNA damage from increased transcription-replication collision events. We report that deregulated transcription following BRD4 loss in cancer cells leads to the accumulation of RNA:DNA hybrids (R-loops) and collisions with the replication machinery causing replication stress and DNA damage. Whole genome BRD4 and γH2AX ChIP-Seq with R-loop IP qPCR reveals that BRD4 inhibition leads to accumulation of R-loops and DNA damage at a subset of known BDR4, JMJD6, and CHD4 co-regulated genes. Interference with BRD4 function causes transcriptional downregulation of the DNA damage response protein TopBP1, resulting in failure to activate the ATR-Chk1 pathway despite increased replication stress, leading to apoptotic cell death in S-phase and mitotic catastrophe. These findings demonstrate that inhibition of BRD4 induces transcription-replication conflicts, DNA damage, and cell death in oncogenic cells.


Assuntos
Proteínas de Ciclo Celular/farmacologia , Dano ao DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Estruturas R-Loop/efeitos dos fármacos , Fatores de Transcrição/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Transporte , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Cromatina , Proteínas de Ligação a DNA , Instabilidade Genômica , Células HeLa , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Neoplasias/terapia , Proteínas Nucleares/metabolismo , Fase S , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma
9.
Nat Commun ; 11(1): 3907, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764578

RESUMO

Nucleic acids can fold into G-quadruplex (G4) structures that can fine-tune biological processes. Proteins are required to recognize G4 structures and coordinate their function. Here we identify Zuo1 as a novel G4-binding protein in vitro and in vivo. In vivo in the absence of Zuo1 fewer G4 structures form, cell growth slows and cells become UV sensitive. Subsequent experiments reveal that these cellular changes are due to reduced levels of G4 structures. Zuo1 function at G4 structures results in the recruitment of nucleotide excision repair (NER) factors, which has a positive effect on genome stability. Cells lacking functional NER, as well as Zuo1, accumulate G4 structures, which become accessible to translesion synthesis. Our results suggest a model in which Zuo1 supports NER function and regulates the choice of the DNA repair pathway nearby G4 structures.


Assuntos
Reparo do DNA/fisiologia , Quadruplex G , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sítios de Ligação/genética , Dano ao DNA , Reparo do DNA/genética , DNA Fúngico/química , DNA Fúngico/genética , DNA Fúngico/metabolismo , Deleção de Genes , Aptidão Genética , Genoma Fúngico , Instabilidade Genômica , Modelos Biológicos , Chaperonas Moleculares/genética , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
10.
Nat Commun ; 11(1): 3940, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769985

RESUMO

R-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops in the human genome, here, we map RNA:DNA hybrids, replication stress markers and DNA double-strand breaks (DSBs) in cells depleted for Topoisomerase I (Top1), an enzyme that relaxes DNA supercoiling and prevents R-loop formation. RNA:DNA hybrids are found at both promoters (TSS) and terminators (TTS) of highly expressed genes. In contrast, the phosphorylation of RPA by ATR is only detected at TTS, which are preferentially replicated in a head-on orientation relative to the direction of transcription. In Top1-depleted cells, DSBs also accumulate at TTS, leading to persistent checkpoint activation, spreading of γ-H2AX on chromatin and global replication fork slowdown. These data indicate that fork pausing at the TTS of highly expressed genes containing R-loops prevents head-on conflicts between replication and transcription and maintains genome integrity in a Top1-dependent manner.


Assuntos
Replicação do DNA , DNA Topoisomerases Tipo I/metabolismo , Estruturas R-Loop/genética , Regiões Terminadoras Genéticas/genética , Transcrição Genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo I/genética , Técnicas de Silenciamento de Genes , Instabilidade Genômica , Células HEK293 , Células HeLa , Humanos , Fosforilação , Regiões Promotoras Genéticas , RNA Interferente Pequeno/metabolismo
11.
Mol Cell ; 79(5): 824-835.e5, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32649882

RESUMO

DNA-protein crosslinks (DPCs) are highly toxic DNA lesions that threaten genomic integrity. Recent findings highlight that SPRTN, a specialized DNA-dependent metalloprotease, is a central player in proteolytic cleavage of DPCs. Previous studies suggest that SPRTN deubiquitination is important for its chromatin association and activation. However, the regulation and consequences of SPRTN deubiquitination remain unclear. Here we report that, in response to DPC induction, the deubiquitinase VCPIP1/VCIP135 is phosphorylated and activated by ATM/ATR. VCPIP1, in turn, deubiquitinates SPRTN and promotes its chromatin relocalization. Deubiquitination of SPRTN is required for its subsequent acetylation, which promotes SPRTN relocation to the site of chromatin damage. Furthermore, Vcpip1 knockout mice are prone to genomic instability and premature aging. We propose a model where two sequential post-translational modifications (PTMs) regulate SPRTN chromatin accessibility to repair DPCs and maintain genomic stability and a healthy lifespan.


Assuntos
Envelhecimento/genética , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Acetilação , Envelhecimento/metabolismo , Animais , Linhagem Celular , Dano ao DNA , Proteínas de Ligação a DNA/genética , Enzimas Desubiquitinantes/metabolismo , Endopeptidases/metabolismo , Feminino , Instabilidade Genômica , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Ubiquitinação
12.
Mol Cell ; 79(5): 836-845.e7, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32649884

RESUMO

The inactive X chromosome (Xi) is inherently susceptible to genomic aberrations. Replication stress (RS) has been proposed as an underlying cause, but the mechanisms that protect from Xi instability remain unknown. Here, we show that macroH2A1.2, an RS-protective histone variant enriched on the Xi, is required for Xi integrity and female survival. Mechanistically, macroH2A1.2 counteracts its structurally distinct and equally Xi-enriched alternative splice variant, macroH2A1.1. Comparative proteomics identified a role for macroH2A1.1 in alternative end joining (alt-EJ), which accounts for Xi anaphase defects in the absence of macroH2A1.2. Genomic instability was rescued by simultaneous depletion of macroH2A1.1 or alt-EJ factors, and mice deficient for both macroH2A1 variants harbor no overt female defects. Notably, macroH2A1 splice variant imbalance affected alt-EJ capacity also in tumor cells. Together, these findings identify macroH2A1 splicing as a modulator of genome maintenance that ensures Xi integrity and may, more broadly, predict DNA repair outcome in malignant cells.


Assuntos
Processamento Alternativo , Reparo do DNA , Epigênese Genética , Instabilidade Genômica , Histonas/fisiologia , Anáfase , Animais , Linhagem Celular , Instabilidade Cromossômica , Cromossomos Humanos X , Feminino , Histonas/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
13.
Proc Natl Acad Sci U S A ; 117(32): 19415-19424, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32719125

RESUMO

Synthetic lethality strategies for cancer therapy exploit cancer-specific genetic defects to identify targets that are uniquely essential to the survival of tumor cells. Here we show RAD27/FEN1, which encodes flap endonuclease 1 (FEN1), a structure-specific nuclease with roles in DNA replication and repair, and has the greatest number of synthetic lethal interactions with Saccharomyces cerevisiae genome instability genes, is a druggable target for an inhibitor-based approach to kill cancers with defects in homologous recombination (HR). The vulnerability of cancers with HR defects to FEN1 loss was validated by studies showing that small-molecule FEN1 inhibitors and FEN1 small interfering RNAs (siRNAs) selectively killed BRCA1- and BRCA2-defective human cell lines. Furthermore, the differential sensitivity to FEN1 inhibition was recapitulated in mice, where a small-molecule FEN1 inhibitor reduced the growth of tumors established from drug-sensitive but not drug-resistant cancer cell lines. FEN1 inhibition induced a DNA damage response in both sensitive and resistant cell lines; however, sensitive cell lines were unable to recover and replicate DNA even when the inhibitor was removed. Although FEN1 inhibition activated caspase to higher levels in sensitive cells, this apoptotic response occurred in p53-defective cells and cell killing was not blocked by a pan-caspase inhibitor. These results suggest that FEN1 inhibitors have the potential for therapeutically targeting HR-defective cancers such as those resulting from BRCA1 and BRCA2 mutations, and other genetic defects.


Assuntos
Antineoplásicos/farmacologia , Endonucleases Flap/antagonistas & inibidores , Recombinação Homóloga/efeitos dos fármacos , Neoplasias/genética , Animais , Proteína BRCA1/deficiência , Proteína BRCA1/genética , Proteína BRCA2/deficiência , Proteína BRCA2/genética , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Endonucleases Flap/genética , Instabilidade Genômica/genética , Humanos , Camundongos , Neoplasias/tratamento farmacológico , RNA Interferente Pequeno/farmacologia , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Mutações Sintéticas Letais , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Proc Natl Acad Sci U S A ; 117(31): 18880-18890, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32694208

RESUMO

Genomic instability contributes to tumorigenesis through the amplification and deletion of cancer driver genes. DNA copy number (CN) profiling of ensembles of tumors allows a thermodynamic analysis of the profile for each tumor. The free energy of the distribution of CNs is found to be a monotonically increasing function of the average chromosomal ploidy. The dependence is universal across several cancer types. Surprisal analysis distinguishes two main known subgroups: tumors with cells that have or have not undergone whole-genome duplication (WGD). The analysis uncovers that CN states having a narrower distribution are energetically more favorable toward the WGD transition. Surprisal analysis also determines the deviations from a fully stable-state distribution. These deviations reflect constraints imposed by tumor fitness selection pressures. The results point to CN changes that are more common in high-ploidy tumors and thus support altered selection pressures upon WGD.


Assuntos
Dosagem de Genes/genética , Instabilidade Genômica/genética , Neoplasias/genética , Variações do Número de Cópias de DNA/genética , Genoma/genética , Humanos , Ploidias , Termodinâmica
15.
Nat Commun ; 11(1): 3613, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32680994

RESUMO

Common fragile sites (CFSs) are regions susceptible to replication stress and are hotspots for chromosomal instability in cancer. Several features were suggested to underlie CFS instability, however, these features are prevalent across the genome. Therefore, the molecular mechanisms underlying CFS instability remain unclear. Here, we explore the transcriptional profile and DNA replication timing (RT) under mild replication stress in the context of the 3D genome organization. The results reveal a fragility signature, comprised of a TAD boundary overlapping a highly transcribed large gene with APH-induced RT-delay. This signature enables precise mapping of core fragility regions in known CFSs and identification of novel fragile sites. CFS stability may be compromised by incomplete DNA replication and repair in TAD boundaries core fragility regions leading to genomic instability. The identified fragility signature will allow for a more comprehensive mapping of CFSs and pave the way for investigating mechanisms promoting genomic instability in cancer.


Assuntos
Sítios Frágeis do Cromossomo/genética , Período de Replicação do DNA/genética , Genoma Humano , Instabilidade Genômica , Afidicolina/farmacologia , Linhagem Celular , Sequenciamento de Cromatina por Imunoprecipitação , Mapeamento Cromossômico/métodos , DNA/química , Período de Replicação do DNA/efeitos dos fármacos , Fibroblastos , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias/genética , Conformação de Ácido Nucleico , Sensibilidade e Especificidade , Transcrição Genética/efeitos dos fármacos
16.
PLoS Genet ; 16(7): e1008611, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32658892

RESUMO

Epimutations in fungal pathogens are emerging as novel phenomena that could explain the fast-developing resistance to antifungal drugs and other stresses. These epimutations are generated by RNA interference (RNAi) mechanisms that transiently silence specific genes to overcome stressful stimuli. The early-diverging fungus Mucor circinelloides exercises a fine control over two interacting RNAi pathways to produce epimutants: the canonical RNAi pathway and a new RNAi degradative pathway. The latter is considered a non-canonical RNAi pathway (NCRIP) because it relies on RNA-dependent RNA polymerases (RdRPs) and a novel ribonuclease III-like named R3B2 to degrade target transcripts. Here in this work, we uncovered the role of NCRIP in regulating virulence processes and transposon movements through key components of the pathway, RdRP1 and R3B2. Mutants in these genes are unable to launch a proper virulence response to macrophage phagocytosis, resulting in a decreased virulence potential. The transcriptomic profile of rdrp1Δ and r3b2Δ mutants revealed a pre-exposure adaptation to the stressful phagosomal environment even when the strains are not confronted by macrophages. These results suggest that NCRIP represses key targets during regular growth and releases its control when a stressful environment challenges the fungus. NCRIP interacts with the RNAi canonical core to protect genome stability by controlling the expression of centromeric retrotransposable elements. In the absence of NCRIP, these retrotransposons are robustly repressed by the canonical RNAi machinery; thus, supporting the antagonistic role of NCRIP in containing the epimutational pathway. Both interacting RNAi pathways might be essential to govern host-pathogen interactions through transient adaptations, contributing to the unique traits of the emerging infection mucormycosis.


Assuntos
Mucorales/genética , Mucormicose/genética , Interferência de RNA , Ribonuclease III/genética , Antifúngicos/farmacologia , Farmacorresistência Fúngica/genética , Epigênese Genética/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/genética , Instabilidade Genômica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Mucorales/patogenicidade , Mucormicose/microbiologia , Mutação/genética , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , Virulência/genética
17.
Nat Commun ; 11(1): 3531, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669601

RESUMO

Homologous recombination (HR) factors were recently implicated in DNA replication fork remodeling and protection. While maintaining genome stability, HR-mediated fork remodeling promotes cancer chemoresistance, by as-yet elusive mechanisms. Five HR cofactors - the RAD51 paralogs RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3 - recently emerged as crucial tumor suppressors. Albeit extensively characterized in DNA repair, their role in replication has not been addressed systematically. Here, we identify all RAD51 paralogs while screening for modulators of RAD51 recombinase upon replication stress. Single-molecule analysis of fork progression and architecture in isogenic cellular systems shows that the BCDX2 subcomplex restrains fork progression upon stress, promoting fork reversal. Accordingly, BCDX2 primes unscheduled degradation of reversed forks in BRCA2-defective cells, boosting genomic instability. Conversely, the CX3 subcomplex is dispensable for fork reversal, but mediates efficient restart of reversed forks. We propose that RAD51 paralogs sequentially orchestrate clinically relevant transactions at replication forks, cooperatively promoting fork remodeling and restart.


Assuntos
Replicação do DNA , Rad51 Recombinase/metabolismo , Proteína BRCA2/metabolismo , Linhagem Celular Tumoral , Estruturas Cromossômicas/metabolismo , Cromossomos/ultraestrutura , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica , Recombinação Homóloga , Humanos , Microscopia , Mutagênicos , Mutação , Osteossarcoma/metabolismo , RNA Interferente Pequeno/metabolismo
18.
PLoS Genet ; 16(7): e1008933, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32692737

RESUMO

Structure-specific endonucleases (SSEs) play key roles in DNA replication, recombination, and repair. SSEs must be tightly regulated to ensure genome stability but their regulatory mechanisms remain incompletely understood. Here, we show that in the fission yeast Schizosaccharomyces pombe, the activities of two SSEs, Dna2 and Rad16 (ortholog of human XPF), are temporally controlled during the cell cycle by the CRL4Cdt2 ubiquitin ligase. CRL4Cdt2 targets Pxd1, an inhibitor of Dna2 and an activator of Rad16, for degradation in S phase. The ubiquitination and degradation of Pxd1 is dependent on CRL4Cdt2, PCNA, and a PCNA-binding degron motif on Pxd1. CRL4Cdt2-mediated Pxd1 degradation prevents Pxd1 from interfering with the normal S-phase functions of Dna2. Moreover, Pxd1 degradation leads to a reduction of Rad16 nuclease activity in S phase, and restrains Rad16-mediated single-strand annealing, a hazardous pathway of repairing double-strand breaks. These results demonstrate a new role of the CRL4Cdt2 ubiquitin ligase in genome stability maintenance and shed new light on how SSE activities are regulated during the cell cycle.


Assuntos
Proteínas de Ligação a DNA/genética , Endonucleases Flap/genética , Proteínas Nucleares/genética , Proteínas de Schizosaccharomyces pombe/genética , Reparo do DNA/genética , Replicação do DNA/genética , Instabilidade Genômica/genética , Humanos , Fase S/genética , Schizosaccharomyces/genética , Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética
19.
Sheng Wu Gong Cheng Xue Bao ; 36(5): 838-848, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32567267

RESUMO

DNA methylation is an epigenetic modification that forms an important regulation mechanism of gene expression in organisms across kingdoms. Aberrant patterns of DNA methylation can lead to plant developmental abnormalities. In this article, we briefly discuss DNA methylation in plants and summarize its functions and biological roles in regulating gene expression and maintaining genomic stability, plant development, as well as plant responses to biotic and abiotic stresses. We intended to provide a concise reference for further understanding of the mechanism of DNA methylation and potential applications of epigenetic manipulation for crop improvement.


Assuntos
Metilação de DNA , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Plantas , Produção Agrícola/tendências , Instabilidade Genômica , Plantas/genética , Pesquisa/tendências , Estresse Fisiológico
20.
PLoS Genet ; 16(6): e1008799, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32502208

RESUMO

TRF2 and TRF1 are a key component in shelterin complex that associates with telomeric DNA and protects chromosome ends. BRM is a core ATPase subunit of SWI/SNF chromatin remodeling complex. Whether and how BRM-SWI/SNF complex is engaged in chromatin end protection by telomeres is unknown. Here, we report that depletion of BRM does not affect heterochromatin state of telomeres, but results in telomere dysfunctional phenomena including telomere uncapping and replication defect. Mechanistically, expression of TRF2 and TRF1 is jointly regulated by BRM-SWI/SNF complex, which is localized to promoter region of both genes and facilitates their transcription. BRM-deficient cells bear increased TRF2-free or TRF1-free telomeres due to insufficient expression. Importantly, BRM depletion-induced telomere uncapping or replication defect can be rescued by compensatory expression of exogenous TRF2 or TRF1, respectively. Together, these results identify a new function of BRM-SWI/SNF complex in enabling functional telomeres for maintaining genome stability.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Telômero/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/genética , Proteína 2 de Ligação a Repetições Teloméricas/genética , Fatores de Transcrição/metabolismo , Instabilidade Genômica , Células HEK293 , Células HeLa , Células Hep G2 , Heterocromatina/metabolismo , Humanos , Regiões Promotoras Genéticas , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Fatores de Transcrição/genética
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