RESUMO
Conflicts between transcription and replication are a potent source of DNA damage. Co-transcriptional R-loops could aggravate such conflicts by creating an additional barrier to replication fork progression. Here, we use a defined episomal system to investigate how conflict orientation and R-loop formation influence genome stability in human cells. R-loops, but not normal transcription complexes, induce DNA breaks and orientation-specific DNA damage responses during conflicts with replication forks. Unexpectedly, the replisome acts as an orientation-dependent regulator of R-loop levels, reducing R-loops in the co-directional (CD) orientation but promoting their formation in the head-on (HO) orientation. Replication stress and deregulated origin firing increase the number of HO collisions leading to genome-destabilizing R-loops. Our findings connect DNA replication to R-loop homeostasis and suggest a mechanistic basis for genome instability resulting from deregulated DNA replication, observed in cancer and other disease states.
Assuntos
Replicação do DNA , Transcrição Gênica , Dano ao DNA , Período de Replicação do DNA , Instabilidade Genômica , Células HEK293 , Humanos , PlasmídeosRESUMO
G-quadruplexes (G4s) form throughout the genome and influence important cellular processes. Their deregulation can challenge DNA replication fork progression and threaten genome stability. Here, we demonstrate an unexpected role for the double-stranded DNA (dsDNA) translocase helicase-like transcription factor (HLTF) in responding to G4s. We show that HLTF, which is enriched at G4s in the human genome, can directly unfold G4s in vitro and uses this ATP-dependent translocase function to suppress G4 accumulation throughout the cell cycle. Additionally, MSH2 (a component of MutS heterodimers that bind G4s) and HLTF act synergistically to suppress G4 accumulation, restrict alternative lengthening of telomeres, and promote resistance to G4-stabilizing drugs. In a discrete but complementary role, HLTF restrains DNA synthesis when G4s are stabilized by suppressing primase-polymerase (PrimPol)-dependent repriming. Together, the distinct roles of HLTF in the G4 response prevent DNA damage and potentially mutagenic replication to safeguard genome stability.
RESUMO
The complex machineries involved in replication and transcription translocate along the same DNA template, often in opposing directions and at different rates. These processes routinely interfere with each other in prokaryotes, and mounting evidence now suggests that RNA polymerase complexes also encounter replication forks in higher eukaryotes. Indeed, cells rely on numerous mechanisms to avoid, tolerate, and resolve such transcription-replication conflicts, and the absence of these mechanisms can lead to catastrophic effects on genome stability and cell viability. In this article, we review the cellular responses to transcription-replication conflicts and highlight how these inevitable encounters shape the genome and impact diverse cellular processes.
Assuntos
Bactérias/metabolismo , Replicação do DNA , Eucariotos/metabolismo , Transcrição Gênica , Animais , Ciclo Celular , Reparo do DNA , RNA Polimerases Dirigidas por DNA/metabolismo , Instabilidade Genômica , HumanosRESUMO
In recent years, increasing evidence has highlighted the profound connection between DNA damage repair and the activation of immune responses. We spoke with researchers about their mechanistic interplays and the implications for cancer and other diseases.
Assuntos
Dano ao DNA , Reparo do DNA , Transdução de Sinais , ImunidadeRESUMO
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/genéticaRESUMO
One way to preserve a rare book is to lock it away from all potential sources of damage. Of course, an inaccessible book is also of little use, and the paper and ink will continue to degrade with age in any case. Like a book, the information stored in our DNA needs to be read, but it is also subject to continuous assault and therefore needs to be protected. In this Review, we examine how the replication stress response that is controlled by the kinase ataxia telangiectasia and Rad3-related (ATR) senses and resolves threats to DNA integrity so that the DNA remains available to read in all of our cells. We discuss the multiple data that have revealed an elegant yet increasingly complex mechanism of ATR activation. This involves a core set of components that recruit ATR to stressed replication forks, stimulate kinase activity and amplify ATR signalling. We focus on the activities of ATR in the control of cell cycle checkpoints, origin firing and replication fork stability, and on how proper regulation of these processes is crucial to ensure faithful duplication of a challenging genome.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Replicação do DNA , Eucariotos/metabolismo , Animais , Genoma , Genoma Humano , Humanos , Transdução de SinaisRESUMO
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/genéticaRESUMO
DNA replication stress can stall replication forks, leading to genome instability. DNA damage tolerance pathways assist fork progression, promoting replication fork reversal, translesion DNA synthesis (TLS), and repriming. In the absence of the fork remodeler HLTF, forks fail to slow following replication stress, but underlying mechanisms and cellular consequences remain elusive. Here, we demonstrate that HLTF-deficient cells fail to undergo fork reversal in vivo and rely on the primase-polymerase PRIMPOL for repriming, unrestrained replication, and S phase progression upon limiting nucleotide levels. By contrast, in an HLTF-HIRAN mutant, unrestrained replication relies on the TLS protein REV1. Importantly, HLTF-deficient cells also exhibit reduced double-strand break (DSB) formation and increased survival upon replication stress. Our findings suggest that HLTF promotes fork remodeling, preventing other mechanisms of replication stress tolerance in cancer cells. This remarkable plasticity of the replication fork may determine the outcome of replication stress in terms of genome integrity, tumorigenesis, and response to chemotherapy.
Assuntos
Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , DNA/biossíntese , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , DNA/genética , Dano ao DNA/genética , DNA Primase/metabolismo , DNA Primase/fisiologia , Reparo do DNA/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/fisiologia , Células HEK293 , Humanos , Células K562 , Enzimas Multifuncionais/metabolismo , Enzimas Multifuncionais/fisiologia , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/fisiologia , Fatores de Transcrição/genéticaRESUMO
During transcription, the nascent RNA strand can base pair with its template DNA, displacing the non-template strand as ssDNA and forming a structure called an R-loop. R-loops are common across many domains of life and cause DNA damage in certain contexts. In this review, we summarize recent results implicating R-loops as important regulators of cellular processes such as transcription termination, gene regulation, and DNA repair. We also highlight recent work suggesting that R-loops can be problematic to cells as blocks to efficient transcription and replication that trigger the DNA damage response. Finally, we discuss how R-loops may contribute to cancer, neurodegeneration, and inflammatory diseases and compare the available next-generation sequencing-based approaches to map R-loops genome wide.
Assuntos
Núcleo Celular/fisiologia , DNA/genética , Genoma , Instabilidade Genômica , Ácidos Nucleicos Heteroduplexes/genética , RNA/genética , Animais , DNA/química , DNA/metabolismo , Dano ao DNA , Reparo do DNA , Regulação da Expressão Gênica , Humanos , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Heteroduplexes/metabolismo , RNA/química , RNA/metabolismo , Relação Estrutura-Atividade , Transcrição GênicaRESUMO
A key property of adult stem cells is their ability to persist in a quiescent state for prolonged periods of time. The quiescent state is thought to contribute to stem cell resilience by limiting accumulation of DNA replicationassociated mutations. Moreover, cellular stress response factors are thought to play a role in maintaining quiescence and stem cell integrity. We utilized muscle stem cells (MuSCs) as a model of quiescent stem cells and find that the replication stress response protein, ATR (Ataxia Telangiectasia and Rad3-Related), is abundant and active in quiescent but not activated MuSCs. Concurrently, MuSCs display punctate RPA (replication protein A) and R-loop foci, both key triggers for ATR activation. To discern the role of ATR in MuSCs, we generated MuSC-specific ATR conditional knockout (ATRcKO) mice. Surprisingly, ATR ablation results in increased MuSC quiescence exit. Phosphoproteomic analysis of ATRcKO MuSCs reveals enrichment of phosphorylated cyclin F, a key component of the Skp1Cul1F-box protein (SCF) ubiquitin ligase complex and regulator of key cell-cycle transition factors, such as the E2F family of transcription factors. Knocking down cyclin F or inhibiting the SCF complex results in E2F1 accumulation and in MuSCs exiting quiescence, similar to ATR-deficient MuSCs. The loss of ATR could be counteracted by inhibiting casein kinase 2 (CK2), the kinase responsible for phosphorylating cyclin F. We propose a model in which MuSCs express cell-cycle progression factors but ATR, in coordination with the cyclin FSCF complex, represses premature stem cell quiescence exit via ubiquitinproteasome degradation of these factors.
Assuntos
Proteínas de Ciclo Celular , Ciclinas , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Ciclinas/genética , Ciclinas/metabolismo , Células-Tronco/metabolismoRESUMO
Stalled replication forks are a critical problem for the cell because they can lead to complex genome rearrangements that underlie cell death and disease. Processes such as DNA damage tolerance and replication fork reversal protect stalled forks from these events. A central mediator of these DNA damage responses in humans is the Rad5-related DNA translocase, HLTF. Here, we present biochemical and structural evidence that the HIRAN domain, an ancient and conserved domain found in HLTF and other DNA processing proteins, is a modified oligonucleotide/oligosaccharide (OB) fold that binds to 3' ssDNA ends. We demonstrate that the HIRAN domain promotes HLTF-dependent fork reversal in vitro through its interaction with 3' ssDNA ends found at forks. Finally, we show that HLTF restrains replication fork progression in cells in a HIRAN-dependent manner. These findings establish a mechanism of HLTF-mediated fork reversal and provide insight into the requirement for distinct fork remodeling activities in the cell.
Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Western Blotting , Linhagem Celular Tumoral , Cristalografia por Raios X , DNA/química , DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Espectroscopia de Ressonância Magnética , Modelos Genéticos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
R-loops, consisting of an RNA-DNA hybrid and displaced single-stranded DNA, are physiological structures that regulate various cellular processes occurring on chromatin. Intriguingly, changes in R-loop dynamics have also been associated with DNA damage accumulation and genome instability; however, the mechanisms underlying R-loop-induced DNA damage remain unknown. Here we demonstrate in human cells that R-loops induced by the absence of diverse RNA processing factors, including the RNA/DNA helicases Aquarius (AQR) and Senataxin (SETX), or by the inhibition of topoisomerase I, are actively processed into DNA double-strand breaks (DSBs) by the nucleotide excision repair endonucleases XPF and XPG. Surprisingly, DSB formation requires the transcription-coupled nucleotide excision repair (TC-NER) factor Cockayne syndrome group B (CSB), but not the global genome repair protein XPC. These findings reveal an unexpected and potentially deleterious role for TC-NER factors in driving R-loop-induced DNA damage and genome instability.
Assuntos
Reparo do DNA , Instabilidade Genômica , Dano ao DNA , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Genoma Humano , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
R-loops are dynamic, co-transcriptional nucleic acid structures that facilitate physiological processes but can also cause DNA damage in certain contexts. Perturbations of transcription or R-loop resolution are expected to change their genomic distribution. Next-generation sequencing approaches to map RNA-DNA hybrids, a component of R-loops, have so far not allowed quantitative comparisons between such conditions. Here, we describe quantitative differential DNA-RNA immunoprecipitation (qDRIP), a method combining synthetic RNA-DNA-hybrid internal standards with high-resolution, strand-specific sequencing. We show that qDRIP avoids biases inherent to read-count normalization by accurately profiling signal in regions unaffected by transcription inhibition in human cells, and by facilitating accurate differential peak calling between conditions. We also use these quantitative comparisons to make the first estimates of the absolute count of RNA-DNA hybrids per cell and their half-lives genome-wide. Finally, we identify a subset of RNA-DNA hybrids with high GC skew which are partially resistant to RNase H. Overall, qDRIP allows for accurate normalization in conditions where R-loops are perturbed and for quantitative measurements that provide previously unattainable biological insights.
Assuntos
DNA/metabolismo , Imunoprecipitação/métodos , Hibridização de Ácido Nucleico , Estruturas R-Loop , RNA/metabolismo , Animais , Linhagem Celular , Drosophila/citologia , Biblioteca Gênica , Genoma , Meia-Vida , Células HeLa , Humanos , Reação em Cadeia da Polimerase , Ribonuclease H , Sonicação , Transcrição GênicaRESUMO
The MRN (MRE11-RAD50-NBS1) complex has been implicated in many aspects of the DNA damage response. It has key roles in sensing and processing DNA double-strand breaks, as well as in activation of ATM (ataxia telangiectasia mutated). We reveal a function for MRN in ATR (ATM- and RAD3-related) activation by using defined ATR-activating DNA structures in Xenopus egg extracts. Strikingly, we demonstrate that MRN is required for recruitment of TOPBP1 to an ATR-activating structure that contains a single-stranded DNA (ssDNA) and a double-stranded DNA (dsDNA) junction and that this recruitment is necessary for phosphorylation of CHK1. We also show that the 911 (RAD9-RAD1-HUS1) complex is not required for TOPBP1 recruitment but is essential for TOPBP1 function. Thus, whereas MRN is required for TOPBP1 recruitment at an ssDNA-to-dsDNA junction, 911 is required for TOPBP1 "activation." These findings provide molecular insights into how ATR is activated.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Proteínas Cromossômicas não Histona/metabolismo , Enzimas Reparadoras do DNA , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Humanos , Proteína Homóloga a MRE11 , Complexos Multiproteicos , Conformação de Ácido Nucleico , Fosforilação , Ligação Proteica , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Transfecção , Proteínas Supressoras de Tumor/genética , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMO
Renal ciliopathies are a leading cause of kidney failure, but their exact etiology is poorly understood. NEK8/NPHP9 is a ciliary kinase associated with two renal ciliopathies in humans and mice, nephronophthisis (NPHP) and polycystic kidney disease. Here, we identify NEK8 as a key effector of the ATR-mediated replication stress response. Cells lacking NEK8 form spontaneous DNA double-strand breaks (DSBs) that further accumulate when replication forks stall, and they exhibit reduced fork rates, unscheduled origin firing, and increased replication fork collapse. NEK8 suppresses DSB formation by limiting cyclin A-associated CDK activity. Strikingly, a mutation in NEK8 that is associated with renal ciliopathies affects its genome maintenance functions. Moreover, kidneys of NEK8 mutant mice accumulate DNA damage, and loss of NEK8 or replication stress similarly disrupts renal cell architecture in a 3D-culture system. Thus, NEK8 is a critical component of the DNA damage response that links replication stress with cystic kidney disorders.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cílios/patologia , Quinases Ciclina-Dependentes/metabolismo , Replicação do DNA/genética , Doenças Renais Policísticas/patologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fase S/fisiologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Técnicas de Cultura de Células , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/genética , Cílios/metabolismo , Quinases Ciclina-Dependentes/genética , Dano ao DNA/genética , Instabilidade Genômica , Humanos , Camundongos , Mutação/genética , Quinases Relacionadas a NIMA , Fosforilação , Doenças Renais Policísticas/metabolismo , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Estresse FisiológicoRESUMO
The DNA damage response kinase ataxia telangiectasia and Rad3-related (ATR) coordinates much of the cellular response to replication stress. The exact mechanisms by which ATR regulates DNA synthesis in conditions of replication stress are largely unknown, but this activity is critical for the viability and proliferation of cancer cells, making ATR a potential therapeutic target. Here we use selective ATR inhibitors to demonstrate that acute inhibition of ATR kinase activity yields rapid cell lethality, disrupts the timing of replication initiation, slows replication elongation, and induces fork collapse. We define the mechanism of this fork collapse, which includes SLX4-dependent cleavage yielding double-strand breaks and CtIP-dependent resection generating excess single-stranded template and nascent DNA strands. Our data suggest that the DNA substrates of these nucleases are generated at least in part by the SMARCAL1 DNA translocase. Properly regulated SMARCAL1 promotes stalled fork repair and restart; however, unregulated SMARCAL1 contributes to fork collapse when ATR is inactivated in both mammalian and Xenopus systems. ATR phosphorylates SMARCAL1 on S652, thereby limiting its fork regression activities and preventing aberrant fork processing. Thus, phosphorylation of SMARCAL1 is one mechanism by which ATR prevents fork collapse, promotes the completion of DNA replication, and maintains genome integrity.
Assuntos
Proteínas de Ciclo Celular/metabolismo , DNA Helicases/metabolismo , Replicação do DNA/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , DNA Helicases/genética , Replicação do DNA/efeitos dos fármacos , DNA de Cadeia Simples/genética , Ativação Enzimática , Humanos , Fosforilação/efeitos dos fármacos , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , XenopusRESUMO
Genome maintenance is a constant concern for cells, and a coordinated response to DNA damage is required to maintain cellular viability and prevent disease. The ataxia-telangiectasia mutated (ATM) and ATM and RAD3-related (ATR) protein kinases act as master regulators of the DNA-damage response by signalling to control cell-cycle transitions, DNA replication, DNA repair and apoptosis. Recent studies have provided new insights into the mechanisms that control ATR activation, have helped to explain the overlapping but non-redundant activities of ATR and ATM in DNA-damage signalling, and have clarified the crucial functions of ATR in maintaining genome integrity.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Instabilidade Genômica/genética , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Biológicos , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de SinaisRESUMO
Studies from Ciccia et al. (2012) and Yuan et al. (2012) in this issue of Molecular Cell, together with Weston et al. (2012), reveal that the translocase ZRANB3/AH2 can recognize K63-linked polyubiquitinated PCNA and plays an important role in restarting stalled replication forks.