RESUMO
In addition to its role as an electron transporter, mitochondrial nicotinamide adenine dinucleotide (NAD+) is an important co-factor for enzymatic reactions, including ADP-ribosylation. Although mitochondria harbor the most intra-cellular NAD+, mitochondrial ADP-ribosylation remains poorly understood. Here we provide evidence for mitochondrial ADP-ribosylation, which was identified using various methodologies including immunofluorescence, western blot, and mass spectrometry. We show that mitochondrial ADP-ribosylation reversibly increases in response to respiratory chain inhibition. Conversely, H2O2-induced oxidative stress reciprocally induces nuclear and reduces mitochondrial ADP-ribosylation. Elevated mitochondrial ADP-ribosylation, in turn, dampens H2O2-triggered nuclear ADP-ribosylation and increases MMS-induced ARTD1 chromatin retention. Interestingly, co-treatment of cells with the mitochondrial uncoupler FCCP decreases PARP inhibitor efficacy. Together, our results suggest that mitochondrial ADP-ribosylation is a dynamic cellular process that impacts nuclear ADP-ribosylation and provide evidence for a NAD+-mediated mitochondrial-nuclear crosstalk.
Assuntos
ADP-Ribosilação , Núcleo Celular/enzimologia , Mitocôndrias/enzimologia , NAD/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , ADP-Ribosilação/efeitos dos fármacos , Animais , Antimicina A/análogos & derivados , Antimicina A/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Cromatina/química , Cromatina/metabolismo , Transporte de Elétrons/efeitos dos fármacos , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Metacrilatos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Mioblastos/enzimologia , Oligomicinas/farmacologia , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/enzimologia , Poli(ADP-Ribose) Polimerase-1/genética , Rotenona/farmacologia , Tiazóis/farmacologiaRESUMO
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/metabolismoRESUMO
The iron-sulfur (FeS) cluster helicase DDX11 is associated with a human disorder termed Warsaw Breakage Syndrome. Interestingly, one disease-associated mutation affects the highly conserved arginine-263 in the FeS cluster-binding motif. Here, we demonstrate that the FeS cluster in DDX11 is required for DNA binding, ATP hydrolysis, and DNA helicase activity, and that arginine-263 affects FeS cluster binding, most likely because of its positive charge. We further show that DDX11 interacts with the replication factors DNA polymerase delta and WDHD1. In vitro, DDX11 can remove DNA obstacles ahead of Pol δ in an ATPase- and FeS domain-dependent manner, and hence generate single-stranded DNA. Accordingly, depletion of DDX11 causes reduced levels of single-stranded DNA, a reduction of chromatin-bound replication protein A, and impaired CHK1 phosphorylation at serine-345. Taken together, we propose that DDX11 plays a role in dismantling secondary structures during DNA replication, thereby promoting CHK1 activation.
Assuntos
Quinase 1 do Ponto de Checagem/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , DNA de Cadeia Simples/biossíntese , Adenosina Trifosfatases/genética , Animais , Proteínas de Ciclo Celular/genética , Quinase 1 do Ponto de Checagem/genética , DNA/química , DNA Polimerase III/química , DNA Polimerase III/genética , Replicação do DNA , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteína de Replicação A/metabolismo , Células Sf9RESUMO
Cellular mechanisms that safeguard genome integrity are often subverted in cancer. To identify cancer-related genome caretakers, we employed a convergent multi-screening strategy coupled to quantitative image-based cytometry and ranked candidate genes according to multivariate readouts reflecting viability, proliferative capacity, replisome integrity, and DNA damage signaling. This unveiled regulators of replication stress resilience, including components of the pre-mRNA cleavage and polyadenylation complex. We show that deregulation of pre-mRNA cleavage impairs replication fork speed and leads to excessive origin activity, rendering cells highly dependent on ATR function. While excessive formation of RNA:DNA hybrids under these conditions was tightly associated with replication-stress-induced DNA damage, inhibition of transcription rescued fork speed, origin activation, and alleviated replication catastrophe. Uncoupling of pre-mRNA cleavage from co-transcriptional processing and export also protected cells from replication-stress-associated DNA damage, suggesting that pre-mRNA cleavage provides a mechanism to efficiently release nascent transcripts and thereby prevent gene gating-associated genomic instability.
Assuntos
Dano ao DNA , Replicação do DNA , Instabilidade Genômica , Neoplasias/genética , Clivagem do RNA , Precursores de RNA/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Transporte Ativo do Núcleo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Poliadenilação , Precursores de RNA/biossíntese , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Proteínas de Ligação a RNARESUMO
The p53-Mdm2 system is key to tumor suppression. We have recently reported that p53 as well as Mdm2 are capable of supporting DNA replication fork progression. On the other hand, we found that Mdm2 is a modifier of chromatin, modulating polycomb repressor complex (PRC)-driven histone modifications. Here we show that, similar to Mdm2 knockdown, the depletion of PRC members impairs DNA synthesis, as determined in fiber assays. In particular, the ubiquitin ligase and PRC1 component RNF2/Ring1B is required to support DNA replication, similar to Mdm2. Moreover, the Ring finger domain of Mdm2 is not only essential for its ubiquitin ligase activity, but also for proper DNA replication. Strikingly, Mdm2 overexpression can rescue RNF2 depletion with regard to DNA replication fork progression, and vice versa, strongly suggesting that the two ubiquitin ligases perform overlapping functions in this context. H2A overexpression also rescues fork progression upon depletion of Mdm2 or RNF2, but only when the ubiquitination sites K118/K119 are present. Depleting the H2A deubiquitinating enzyme BAP1 reduces the fork rate, suggesting that both ubiquitination and deubiquitination of H2A are required to support fork progression. The depletion of Mdm2 elicits the accumulation of RNA/DNA hybrids, suggesting R-loop formation as a mechanism of impaired DNA replication. Accordingly, RNase H overexpression or the inhibition of the transcription elongation kinase CDK9 each rescues DNA replication upon depletion of Mdm2 or RNF2. Taken together, our results suggest that chromatin modification by Mdm2 and PRC1 ensures smooth DNA replication through the avoidance of R-loop formation.
Assuntos
Cromatina/metabolismo , Replicação do DNA , DNA/genética , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA/genética , Linhagem Celular Tumoral , Cromatina/genética , DNA/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Complexo Repressor Polycomb 1/genética , Domínios Proteicos , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/genética , RNA/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , UbiquitinaçãoRESUMO
Exploiting the full potential of anti-cancer drugs necessitates a detailed understanding of their cytotoxic effects. While standard omics approaches are limited to cell population averages, emerging single cell techniques currently lack throughput and are not applicable for compound screens. Here, we employed a versatile and sensitive high-content microscopy-based approach to overcome these limitations and quantify multiple parameters of cytotoxicity at the single cell level and in a cell cycle resolved manner. Applied to PARP inhibitors (PARPi) this approach revealed an S-phase-specific DNA damage response after only 15 min, quantitatively differentiated responses to several clinically important PARPi, allowed for cell cycle resolved analyses of PARP trapping, and predicted conditions of PARPi hypersensitivity and resistance. The approach illuminates cellular mechanisms of drug synergism and, through a targeted multivariate screen, could identify a functional interaction between PARPi olaparib and NEDD8/SCF inhibition, which we show is dependent on PARP1 and linked to PARP1 trapping.
Assuntos
Resistência a Medicamentos/efeitos dos fármacos , Microscopia de Fluorescência/métodos , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Dano ao DNA , Reparo do DNA , Resistência a Medicamentos/genética , Humanos , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Interferência de RNA , Imagem com Lapso de Tempo/métodosRESUMO
The bivalent histone modification reader 53BP1 accumulates around DNA double-strand breaks (DSBs), where it dictates repair pathway choice decisions by limiting DNA end resection. How this function is regulated locally and across the cell cycle to channel repair reactions toward non-homologous end joining (NHEJ) in G1 and promote homology-directed repair (HDR) in S/G2 is insufficiently understood. Here, we show that the ability of 53BP1 to accumulate around DSBs declines as cells progress through S phase and reveal that the inverse relationship between 53BP1 recruitment and replicated chromatin is linked to the replication-coupled dilution of 53BP1's target mark H4K20me2. Consistently, premature maturation of post-replicative chromatin restores H4K20me2 and rescues 53BP1 accumulation on replicated chromatin. The H4K20me2-mediated chromatin association of 53BP1 thus represents an inbuilt mechanism to distinguish DSBs in pre- versus post-replicative chromatin, allowing for localized repair pathway choice decisions based on the availability of replication-generated template strands for HDR.
Assuntos
Cromatina/metabolismo , Replicação do DNA , Histonas/metabolismo , Lisina/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Humanos , Modelos Biológicos , Reparo de DNA por RecombinaçãoRESUMO
Harmful oxidation of proteins, lipids and nucleic acids is observed when reactive oxygen species (ROS) are produced excessively and/or the antioxidant capacity is reduced, causing 'oxidative stress'. Nuclear poly-ADP-ribose (PAR) formation is thought to be induced in response to oxidative DNA damage and to promote cell death under sustained oxidative stress conditions. However, what exactly triggers PAR induction in response to oxidative stress is incompletely understood. Using reverse phase protein array (RPPA) and in-depth analysis of key stress signaling components, we observed that PAR formation induced by H2O2 was mediated by the PLC/IP3R/Ca(2+)/PKCα signaling axis. Mechanistically, H2O2-induced PAR formation correlated with Ca(2+)-dependent DNA damage, which, however, was PKCα-independent. In contrast, PAR formation was completely lost upon knockdown of PKCα, suggesting that DNA damage alone was not sufficient for inducing PAR formation, but required a PKCα-dependent process. Intriguingly, the loss of PAR formation observed upon PKCα depletion was overcome when the chromatin structure-modifying protein HMGB1 was co-depleted with PKCα, suggesting that activation and nuclear translocation of PKCα releases the inhibitory effect of HMGB1 on PAR formation. Together, these results identify PKCα and HMGB1 as important co-regulators involved in H2O2-induced PAR formation, a finding that may have important relevance for oxidative stress-associated pathophysiological conditions.
Assuntos
Proteína HMGB1/metabolismo , Peróxido de Hidrogênio/farmacologia , Poli Adenosina Difosfato Ribose/metabolismo , Proteína Quinase C-alfa/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/enzimologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Cromatina/metabolismo , Quebras de DNA/efeitos dos fármacos , DNA Glicosilases/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Histonas/metabolismo , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Camundongos , Células NIH 3T3 , Fosforilação/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fosfolipases Tipo C/metabolismoRESUMO
The human genome encodes 45 kinesin motor proteins that drive cell division, cell motility, intracellular trafficking and ciliary function. Determining the cellular function of each kinesin would benefit from specific small-molecule inhibitors. However, screens have yielded only a few specific inhibitors. Here we present a novel chemical-genetic approach to engineer kinesin motors that can carry out the function of the wild-type motor yet can also be efficiently inhibited by small, cell-permeable molecules. Using kinesin-1 as a prototype, we develop two independent strategies to generate inhibitable motors, and characterize the resulting inhibition in single-molecule assays and in cells. We further apply these two strategies to create analogously inhibitable kinesin-3 motors. These inhibitable motors will be of great utility to study the functions of specific kinesins in a dynamic manner in cells and animals. Furthermore, these strategies can be used to generate inhibitable versions of any motor protein of interest.
Assuntos
Cinesinas/antagonistas & inibidores , Microtúbulos/efeitos dos fármacos , Engenharia de Proteínas , Bibliotecas de Moléculas Pequenas/farmacologia , Moduladores de Tubulina/farmacologia , Animais , Células COS , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Chlorocebus aethiops , Drosophila melanogaster , Dineínas/genética , Dineínas/metabolismo , Expressão Gênica , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Camundongos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Imagem Molecular , Miosinas/genética , Miosinas/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Transporte Proteico , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Transfecção , Moduladores de Tubulina/síntese químicaRESUMO
Embryonic stem cells (ESCs) represent a transient biological state, where pluripotency is coupled with fast proliferation. ESCs display a constitutively active DNA damage response (DDR), but its molecular determinants have remained elusive. Here we show in cultured ESCs and mouse embryos that H2AX phosphorylation is dependent on Ataxia telangiectasia and Rad3 related (ATR) and is associated with chromatin loading of the ssDNA-binding proteins RPA and RAD51. Single-molecule analysis of replication intermediates reveals massive ssDNA gap accumulation, reduced fork speed and frequent fork reversal. All these marks of replication stress do not impair the mitotic process and are rapidly lost at differentiation onset. Delaying the G1/S transition in ESCs allows formation of 53BP1 nuclear bodies and suppresses ssDNA accumulation, fork slowing and reversal in the following S-phase. Genetic inactivation of fork slowing and reversal leads to chromosomal breakage in unperturbed ESCs. We propose that rapid cell cycle progression makes ESCs dependent on effective replication-coupled mechanisms to protect genome integrity.
Assuntos
Dano ao DNA , Replicação do DNA , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular , Fase G1 , Células-Tronco Embrionárias Murinas/metabolismo , Rad51 Recombinase/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Blastocisto/metabolismo , Western Blotting , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Eletroforese em Gel de Campo Pulsado , Citometria de Fluxo , Histonas/metabolismo , Camundongos , Microscopia Confocal , Microscopia Eletrônica , Microscopia de Fluorescência , Mitose , Mórula/metabolismo , Fosforilação , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína de Replicação A/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
Post-translational modifications (PTMs) regulate many aspects of protein function and are indispensable for the spatio-temporal regulation of cellular processes. The proteome-wide identification of PTM targets has made significant progress in recent years, as has the characterization of their writers, readers, modifiers and erasers. One of the most elusive PTMs is poly(ADP-ribosyl)ation (PARylation), a nucleic acid-like PTM involved in chromatin dynamics, genome stability maintenance, transcription, cell metabolism and development. In this article, we provide an overview on our current understanding of the writers of this modification and their targets, as well as the enzymes that degrade and thereby modify and erase poly(ADP-ribose) (PAR). Since many cellular functions of PARylation are exerted through dynamic interactions of PAR-binding proteins with PAR, we discuss the readers of this modification and provide a synthesis of recent findings, which suggest that multiple structurally highly diverse reader modules, ranging from completely folded PAR-binding domains to intrinsically disordered sequence stretches, evolved as PAR effectors to carry out specific cellular functions.
Assuntos
Poli Adenosina Difosfato Ribose/metabolismo , Humanos , Neoplasias/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Intrinsically disordered proteins can phase separate from the soluble intracellular space, and tend to aggregate under pathological conditions. The physiological functions and molecular triggers of liquid demixing by phase separation are not well understood. Here we show in vitro and in vivo that the nucleic acid-mimicking biopolymer poly(ADP-ribose) (PAR) nucleates intracellular liquid demixing. PAR levels are markedly induced at sites of DNA damage, and we provide evidence that PAR-seeded liquid demixing results in rapid, yet transient and fully reversible assembly of various intrinsically disordered proteins at DNA break sites. Demixing, which relies on electrostatic interactions between positively charged RGG repeats and negatively charged PAR, is amplified by aggregation-prone prion-like domains, and orchestrates the earliest cellular responses to DNA breakage. We propose that PAR-seeded liquid demixing is a general mechanism to dynamically reorganize the soluble nuclear space with implications for pathological protein aggregation caused by derailed phase separation.