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1.
Nucleic Acids Res ; 44(3): 1161-78, 2016 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-26578593

RESUMO

Although defective repair of DNA double-strand breaks (DSBs) leads to neurodegenerative diseases, the processes underlying their production and signaling in non-replicating cells are largely unknown. Stabilized topoisomerase I cleavage complexes (Top1cc) by natural compounds or common DNA alterations are transcription-blocking lesions whose repair depends primarily on Top1 proteolysis and excision by tyrosyl-DNA phosphodiesterase-1 (TDP1). We previously reported that stabilized Top1cc produce transcription-dependent DSBs that activate ATM in neurons. Here, we use camptothecin (CPT)-treated serum-starved quiescent cells to induce transcription-blocking Top1cc and show that those DSBs are generated during Top1cc repair from Top1 peptide-linked DNA single-strand breaks generated after Top1 proteolysis and before excision by TDP1. Following DSB induction, ATM activates DNA-PK whose inhibition suppresses H2AX and H2A ubiquitination and the later assembly of activated ATM into nuclear foci. Inhibition of DNA-PK also reduces Top1 ubiquitination and proteolysis as well as resumption of RNA synthesis suggesting that DSB signaling further enhances Top1cc repair. Finally, we show that co-transcriptional DSBs kill quiescent cells. Together, these new findings reveal that DSB production and signaling by transcription-blocking Top1 lesions impact on non-replicating cell fate and provide insights on the molecular pathogenesis of neurodegenerative diseases such as SCAN1 and AT syndromes, which are caused by TDP1 and ATM deficiency, respectively.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA Topoisomerases Tipo I/metabolismo , Proteína Quinase Ativada por DNA/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Camptotecina/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Meios de Cultura Livres de Soro/farmacologia , Quebras de DNA de Cadeia Simples , DNA Topoisomerases Tipo I/genética , Proteína Quinase Ativada por DNA/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Immunoblotting , Microscopia de Fluorescência , Proteínas Nucleares/genética , Interferência de RNA , Transdução de Sinais , Inibidores da Topoisomerase I/farmacologia , Ubiquitinação/efeitos dos fármacos
2.
Cell Rep ; 43(5): 114214, 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38761375

RESUMO

TDP1 removes transcription-blocking topoisomerase I cleavage complexes (TOP1ccs), and its inactivating H493R mutation causes the neurodegenerative syndrome SCAN1. However, the molecular mechanism underlying the SCAN1 phenotype is unclear. Here, we generate human SCAN1 cell models using CRISPR-Cas9 and show that they accumulate TOP1ccs along with changes in gene expression and genomic distribution of R-loops. SCAN1 cells also accumulate transcriptional DNA double-strand breaks (DSBs) specifically in the G1 cell population due to increased DSB formation and lack of repair, both resulting from abortive removal of transcription-blocking TOP1ccs. Deficient TDP1 activity causes increased DSB production, and the presence of mutated TDP1 protein hampers DSB repair by a TDP2-dependent backup pathway. This study provides powerful models to study TDP1 functions under physiological and pathological conditions and unravels that a gain of function of the mutated TDP1 protein, which prevents DSB repair, rather than a loss of TDP1 activity itself, could contribute to SCAN1 pathogenesis.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Mutação , Doenças Neurodegenerativas , Diester Fosfórico Hidrolases , Humanos , Diester Fosfórico Hidrolases/metabolismo , Diester Fosfórico Hidrolases/genética , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Mutação/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo I/genética , Transcrição Gênica , Estruturas R-Loop , Sistemas CRISPR-Cas/genética
3.
Nat Genet ; 55(10): 1721-1734, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37735199

RESUMO

The single-stranded DNA cytosine-to-uracil deaminase APOBEC3B is an antiviral protein implicated in cancer. However, its substrates in cells are not fully delineated. Here APOBEC3B proteomics reveal interactions with a surprising number of R-loop factors. Biochemical experiments show APOBEC3B binding to R-loops in cells and in vitro. Genetic experiments demonstrate R-loop increases in cells lacking APOBEC3B and decreases in cells overexpressing APOBEC3B. Genome-wide analyses show major changes in the overall landscape of physiological and stimulus-induced R-loops with thousands of differentially altered regions, as well as binding of APOBEC3B to many of these sites. APOBEC3 mutagenesis impacts genes overexpressed in tumors and splice factor mutant tumors preferentially, and APOBEC3-attributed kataegis are enriched in RTCW motifs consistent with APOBEC3B deamination. Taken together with the fact that APOBEC3B binds single-stranded DNA and RNA and preferentially deaminates DNA, these results support a mechanism in which APOBEC3B regulates R-loops and contributes to R-loop mutagenesis in cancer.


Assuntos
Neoplasias , Estruturas R-Loop , Humanos , DNA de Cadeia Simples/genética , Estudo de Associação Genômica Ampla , Mutagênese , Neoplasias/genética , Neoplasias/patologia , Citidina Desaminase/genética , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo
4.
Nat Commun ; 13(1): 2961, 2022 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-35618715

RESUMO

RNase H2 is a specialized enzyme that degrades RNA in RNA/DNA hybrids and deficiency of this enzyme causes a severe neuroinflammatory disease, Aicardi Goutières syndrome (AGS). However, the molecular mechanism underlying AGS is still unclear. Here, we show that RNase H2 is associated with a subset of genes, in a transcription-dependent manner where it interacts with RNA Polymerase II. RNase H2 depletion impairs transcription leading to accumulation of R-loops, structures that comprise RNA/DNA hybrids and a displaced DNA strand, mainly associated with short and intronless genes. Importantly, accumulated R-loops are processed by XPG and XPF endonucleases which leads to DNA damage and activation of the immune response, features associated with AGS. Consequently, we uncover a key role for RNase H2 in the transcription of human genes by maintaining R-loop homeostasis. Our results provide insight into the mechanistic contribution of R-loops to AGS pathogenesis.


Assuntos
Estruturas R-Loop , Ribonucleases , Doenças Autoimunes do Sistema Nervoso , DNA/química , Quebras de DNA , Endorribonucleases/metabolismo , Humanos , Inflamação/genética , Malformações do Sistema Nervoso , Estruturas R-Loop/genética , RNA/química , Ribonuclease H/metabolismo , Ribonuclease Pancreático/metabolismo , Ribonucleases/metabolismo
5.
Int Rev Cell Mol Biol ; 364: 195-240, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34507784

RESUMO

Transcription is an essential cellular process but also a major threat to genome integrity. Transcription-associated DNA breaks are particularly detrimental as their defective repair can induce gene mutations and oncogenic chromosomal translocations, which are hallmarks of cancer. The past few years have revealed that transcriptional breaks mainly originate from DNA topological problems generated by the transcribing RNA polymerases. Defective removal of transcription-induced DNA torsional stress impacts on transcription itself and promotes secondary DNA structures, such as R-loops, which can induce DNA breaks and genome instability. Paradoxically, as they relax DNA during transcription, topoisomerase enzymes introduce DNA breaks that can also endanger genome integrity. Stabilization of topoisomerases on chromatin by various anticancer drugs or by DNA alterations, can interfere with transcription machinery and cause permanent DNA breaks and R-loops. Here, we review the role of transcription in mediating DNA breaks, and discuss how deregulation of topoisomerase activity can impact on transcription and DNA break formation, and its connection with cancer.


Assuntos
Quebras de DNA , DNA/química , Neoplasias/genética , Neoplasias/patologia , Transcrição Gênica , Animais , DNA Topoisomerases Tipo I/metabolismo , Instabilidade Genômica , Humanos , Neoplasias/enzimologia
6.
Mol Cell Oncol ; 7(2): 1691905, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32158914

RESUMO

Accumulation of DNA damage in resting cells is an emerging cause of human disease. We identified a mechanism of DNA double-strand break (DSB) formation in non-replicating cells, which strictly depends on transcription. These transcriptional DSBs arise from the twinned processing of R-loops and topoisomerase I and may underlie neurological disorders and cancers.

7.
Nat Genet ; 52(1): 48-55, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31844323

RESUMO

R-loops are nucleic acid structures formed by an RNA:DNA hybrid and unpaired single-stranded DNA that represent a source of genomic instability in mammalian cells1-4. Here we show that N6-methyladenosine (m6A) modification, contributing to different aspects of messenger RNA metabolism5,6, is detectable on the majority of RNA:DNA hybrids in human pluripotent stem cells. We demonstrate that m6A-containing R-loops accumulate during G2/M and are depleted at G0/G1 phases of the cell cycle, and that the m6A reader promoting mRNA degradation, YTHDF2 (ref. 7), interacts with R-loop-enriched loci in dividing cells. Consequently, YTHDF2 knockout leads to increased R-loop levels, cell growth retardation and accumulation of γH2AX, a marker for DNA double-strand breaks, in mammalian cells. Our results suggest that m6A regulates accumulation of R-loops, implying a role for this modification in safeguarding genomic stability.


Assuntos
Adenosina/análogos & derivados , DNA/química , Instabilidade Genômica , Células-Tronco Pluripotentes/metabolismo , Estabilidade de RNA/efeitos dos fármacos , Proteínas de Ligação a RNA/fisiologia , RNA/química , Adenosina/farmacologia , Animais , DNA/efeitos dos fármacos , DNA/genética , Dano ao DNA , Humanos , Camundongos , Camundongos Knockout , Mitose , Células-Tronco Pluripotentes/citologia , RNA/efeitos dos fármacos , RNA/genética , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
8.
Cell Rep ; 28(12): 3167-3181.e6, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31533039

RESUMO

Although accumulation of DNA damage and genomic instability in resting cells can cause neurodegenerative disorders, our understanding of how transcription produces DNA double-strand breaks (DSBs) is limited. Transcription-blocking topoisomerase I cleavage complexes (TOP1ccs) are frequent events that prime DSB production in non-replicating cells. Here, we report a mechanism of their formation by showing that they arise from two nearby single-strand breaks (SSBs) on opposing DNA strands: one SSB from the removal of transcription-blocking TOP1ccs by the TDP1 pathway and the other from the cleavage of R-loops by endonucleases, including XPF, XPG, and FEN1. Genetic defects in TOP1cc removal (TDP1, PNKP, and XRCC1) or in the resolution of R-loops (SETX) enhance DSB formation and prevent their repair. Such deficiencies cause neurological disorders. Owing to the high frequency of TOP1cc trapping and the widespread distribution of R-loops, these persistent transcriptional DSBs could accumulate over time in neuronal cells, contributing to the neurodegenerative diseases.


Assuntos
Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , DNA Topoisomerases Tipo I/metabolismo , Estruturas R-Loop , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Endonucleases Flap/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo
9.
Cell Rep ; 23(6): 1891-1905, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29742442

RESUMO

R-loops comprise an RNA/DNA hybrid and displaced single-stranded DNA. They play important biological roles and are implicated in pathology. Even so, proteins recognizing these structures are largely undefined. Using affinity purification with the S9.6 antibody coupled to mass spectrometry, we defined the RNA/DNA hybrid interactome in HeLa cells. This consists of known R-loop-associated factors SRSF1, FACT, and Top1, and yet uncharacterized interactors, including helicases, RNA processing, DNA repair, and chromatin factors. We validate specific examples of these interactors and characterize their involvement in R-loop biology. A top candidate DHX9 helicase promotes R-loop suppression and transcriptional termination. DHX9 interacts with PARP1, and both proteins prevent R-loop-associated DNA damage. DHX9 and other interactome helicases are overexpressed in cancer, linking R-loop-mediated DNA damage and disease. Our RNA/DNA hybrid interactome provides a powerful resource to study R-loop biology in health and disease.


Assuntos
RNA Helicases DEAD-box/metabolismo , Dano ao DNA , DNA/metabolismo , Proteínas de Neoplasias/metabolismo , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/metabolismo , RNA/metabolismo , Terminação da Transcrição Genética , Camptotecina/farmacologia , Células HEK293 , Células HeLa , Humanos , Imunoprecipitação , Reprodutibilidade dos Testes , Terminação da Transcrição Genética/efeitos dos fármacos
10.
Cell Death Dis ; 9(9): 931, 2018 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-30209297

RESUMO

RHO GTPases regulate essential functions such as the organization of the actin cytoskeleton. The classic members cycle between an active GTP-bound and an inactive GDP-bound conformation whereas atypical members are predominantly GTP-bound. Besides their well-established role, the classic RHO GTPases RHOB and RAC1, are rapidly induced and/or activated by genotoxic stress and contribute to the DNA damage response. Here we used camptothecin, a selective topoisomerase I (TOP1) inhibitor that stabilizes TOP1 cleavage complexes (TOP1cc), to search for other potential early DNA damage-inducible RHO GTPase genes. We identified that an atypical RHO GTPase, RND1, is rapidly induced by camptothecin. RND1 induction is closely associated with the presence of TOP1cc induced by camptothecin or by DNA lesions that elevate TOP1cc levels such as UV and hydrogen peroxide. We further demonstrated that camptothecin increases RND1 gene transcription and mRNA stability. Camptothecin also increases poly(ADP-ribose) polymerase 1 (PARP-1) activity, whose inhibition reduces RND1 transcription. In addition, overexpression of RND1 increases PARP-1, suggesting a cross-talk between PARP-1 and RND1. Finally, RND1 protects cells against camptothecin-induced apoptosis, and hence favors cellular resistance to camptothecin. Together, these findings highlight RND1 as an atypical RHO GTPase early induced by TOP1cc, and show that the TOP1cc-PARP-1-RND1 pathway protects cells against apoptosis induced by camptothecin.


Assuntos
Camptotecina/farmacologia , DNA Topoisomerases Tipo I/genética , DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Poli(ADP-Ribose) Polimerase-1/genética , Transcrição Gênica/genética , Proteínas rho de Ligação ao GTP/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Células HCT116 , Humanos , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/genética , Camundongos , Células NIH 3T3 , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores da Topoisomerase I/farmacologia
11.
J Mol Biol ; 429(21): 3181-3195, 2017 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-27771483

RESUMO

R-loops comprise an RNA/DNA hybrid and a displaced single-stranded DNA. They play crucial biological functions and are implicated in neurological diseases, including ataxias, amyotrophic lateral sclerosis, nucleotide expansion disorders (Friedreich ataxia and fragile X syndrome), and cancer. Currently, it is unclear which mechanisms cause R-loop structures to become pathogenic. The RNA/DNA helicase senataxin (SETX) is one of the best characterised R-loop-binding factors in vivo. Mutations in SETX are linked to two neurodegenerative disorders: ataxia with oculomotor apraxia type 2 (AOA2) and amyotrophic lateral sclerosis type 4 (ALS4). SETX is known to play a role in transcription, neurogenesis, and antiviral response. Here, we review the causes of R-loop dysregulation in neurodegenerative diseases and how these structures contribute to pathomechanisms. We will discuss the importance of SETX as a genome guardian in suppressing aberrant R-loop formation and analyse how SETX mutations can lead to neurodegeneration in AOA2/ALS4. Finally, we will discuss the implications for other R-loop-associated neurodegenerative diseases and point to future therapeutic approaches to treat these disorders.


Assuntos
Regulação da Expressão Gênica , Doenças Neurodegenerativas/genética , RNA Helicases/genética , Transcrição Gênica , DNA Helicases , Humanos , Enzimas Multifuncionais
12.
PLoS One ; 11(1): e0147053, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26784695

RESUMO

Topoisomerase I-DNA-cleavage complexes (Top1cc) stabilized by camptothecin (CPT) have specific effects at transcriptional levels. We recently reported that Top1cc increase antisense transcript (aRNAs) levels at divergent CpG-island promoters and, transiently, DNA/RNA hybrids (R-loop) in nuclear and mitochondrial genomes of colon cancer HCT116 cells. However, the relationship between R-loops and aRNAs was not established. Here, we show that aRNAs can form R-loops in N-TERA-2 cells under physiological conditions, and that promoter-associated R-loops are somewhat increased and extended in length immediately upon cell exposure to CPT. In contrast, persistent Top1ccs reduce the majority of R-loops suggesting that CPT-accumulated aRNAs are not commonly involved in R-loops. The enhancement of aRNAs by Top1ccs is present both in human colon cancer HCT116 cells and WI38 fibroblasts suggesting a common response of cancer and normal cells. Although Top1ccs lead to DSB and DDR kinases activation, we do not detect a dependence of aRNA accumulation on ATM or DNA-PK activation. However, we showed that the cell response to persistent Top1ccs can involve an impairment of aRNA turnover rather than a higher synthesis rate. Finally, a genome-wide analysis shows that persistent Top1ccs also determine an accumulation of sense transcripts at 5'-end gene regions suggesting an increased occurrence of truncated transcripts. Taken together, the results indicate that Top1 may regulate transcription initiation by modulating RNA polymerase-generated negative supercoils, which can in turn favor R-loop formation at promoters, and that transcript accumulation at TSS is a response to persistent transcriptional stress by Top1 poisoning.


Assuntos
Camptotecina/farmacologia , Replicação do DNA , DNA Topoisomerases Tipo I/química , Regiões Promotoras Genéticas/genética , Inibidores da Topoisomerase I/farmacologia , DNA Topoisomerases Tipo I/genética , Células HCT116 , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Gênica
13.
Mol Cell Biol ; 34(16): 3144-55, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24912678

RESUMO

Unlike other Rho GTPases, RhoB is rapidly induced by DNA damage, and its expression level decreases during cancer progression. Because inefficient repair of DNA double-strand breaks (DSBs) can lead to cancer, we investigated whether camptothecin, an anticancer drug that produces DSBs, induces RhoB expression and examined its role in the camptothecin-induced DNA damage response. We show that in camptothecin-treated cells, DSBs induce RhoB expression by a mechanism that depends notably on Chk2 and its substrate HuR, which binds to RhoB mRNA and protects it against degradation. RhoB-deficient cells fail to dephosphorylate γH2AX following camptothecin removal and show reduced efficiency of DSB repair by homologous recombination. These cells also show decreased activity of protein phosphatase 2A (PP2A), a phosphatase for γH2AX and other DNA damage and repair proteins. Thus, we propose that DSBs activate a Chk2-HuR-RhoB pathway that promotes PP2A-mediated dephosphorylation of γH2AX and DSB repair. Finally, we show that RhoB-deficient cells accumulate endogenous γH2AX and chromosomal abnormalities, suggesting that RhoB loss increases DSB-mediated genomic instability and tumor progression.


Assuntos
Quebras de DNA de Cadeia Dupla , Histonas/metabolismo , Proteína Fosfatase 2/metabolismo , Proteína rhoB de Ligação ao GTP/genética , Animais , Antineoplásicos Fitogênicos/farmacologia , Camptotecina/farmacologia , Linhagem Celular Tumoral , Quinase do Ponto de Checagem 2/metabolismo , Aberrações Cromossômicas , Reparo do DNA/genética , Proteínas ELAV/metabolismo , Instabilidade Genômica/genética , Células HCT116 , Humanos , Camundongos , Camundongos Knockout , Fosforilação , Ligação Proteica/genética , Proteína Fosfatase 2/genética , Interferência de RNA , RNA Interferente Pequeno , Proteínas de Ligação a RNA/metabolismo , Inibidores da Topoisomerase I/farmacologia , Proteína rhoB de Ligação ao GTP/biossíntese
14.
J Invest Dermatol ; 134(1): 203-212, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23792460

RESUMO

The role of UVB-induced apoptosis in the formation of squamous cell carcinoma (SCC) is recognized. We previously identified the small RhoB (Ras homolog gene family, member B) GTPase, an early response gene to cellular stress, as a critical protein controlling apoptosis of human keratinocytes after UVB exposure. Here we generated SKH1 (hairless immunocompetent mouse) mice invalidated for RhoB to evaluate its role in UVB-induced skin carcinogenesis in vivo. We show that rhob-/- mice have a lower risk of developing UVB-induced keratotic tumors and actinic keratosis that is associated with a higher sensitivity of UVB-exposed keratinocytes to apoptosis. We extend this observation to primary cultures of normal human keratinocytes in which RhoB was downregulated with small interfering RNA (siRNA) and further show that the hypersensitivity to apoptosis depends on B-cell lymphoma 2 (Bcl-2) downregulation. In rhob-/- mice, the UVB-induced tumors were preferentially undifferentiated and highly proliferative. Finally, we show in humans an almost constant loss of RhoB expression in undifferentiated SCCs. These undifferentiated and RhoB-deficient tumors have elevated phosphorylated histone H2AX (γH2AX) and 53BP1, two markers of DNA double-strand breaks. Together, our results indicate that UVB-induced RhoB expression participates in in vivo SCC initiation by increasing keratinocyte survival. Conversely, RhoB may limit tumor aggressiveness as loss of RhoB expression in tumor cells is associated with tumor progression.


Assuntos
Apoptose/fisiologia , Carcinoma de Células Escamosas/patologia , Queratinócitos/patologia , Neoplasias Cutâneas/patologia , Proteína rhoB de Ligação ao GTP/fisiologia , Animais , Apoptose/genética , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Células Cultivadas , Feminino , Humanos , Queratinócitos/efeitos da radiação , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Pelados , Camundongos Knockout , RNA Interferente Pequeno/genética , Raios Ultravioleta/efeitos adversos , Proteína rhoB de Ligação ao GTP/genética
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