RESUMO
Protein ubiquitylation and sumoylation play key roles in regulating cellular responses to DNA double-strand breaks (DSBs). Here, we show that human RNF4, a small ubiquitin-like modifier (SUMO)-targeted ubiquitin E3 ligase, is recruited to DSBs in a manner requiring its SUMO interaction motifs, the SUMO E3 ligases PIAS1 and PIAS4, and various DSB-responsive proteins. Furthermore, we reveal that RNF4 depletion impairs ubiquitin adduct formation at DSB sites and causes persistent histone H2AX phosphorylation (γH2AX) associated with defective DSB repair, hypersensitivity toward DSB-inducing agents, and delayed recovery from radiation-induced cell cycle arrest. We establish that RNF4 regulates turnover of the DSB-responsive factors MDC1 and replication protein A (RPA) at DNA damage sites and that RNF4-depleted cells fail to effectively replace RPA by the homologous recombination factors BRCA2 and RAD51 on resected DNA. Consistent with previous data showing that RNF4 targets proteins to the proteasome, we show that the proteasome component PSMD4 is recruited to DNA damage sites in a manner requiring its ubiquitin-interacting domains, RNF4 and RNF8. Finally, we establish that PSMD4 binds MDC1 and RPA1 in a DNA damage-induced, RNF4-dependent manner and that PSMD4 depletion cause MDC1 and γH2AX persistence in irradiated cells. RNF4 thus operates as a DSB response factor at the crossroads between the SUMO and ubiquitin systems.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , DNA de Cadeia Simples/metabolismo , Histonas/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Rad51 Recombinase/metabolismo , Proteína de Replicação A/metabolismo , Transativadores/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
In model organisms, classical genetic screening via random mutagenesis provides key insights into the molecular bases of genetic interactions, helping to define synthetic lethality, synthetic viability and drug-resistance mechanisms. The limited genetic tractability of diploid mammalian cells, however, precludes this approach. Here, we demonstrate the feasibility of classical genetic screening in mammalian systems by using haploid cells, chemical mutagenesis and next-generation sequencing, providing a new tool to explore mammalian genetic interactions.
Assuntos
Testes Genéticos , Genoma/efeitos dos fármacos , Genoma/genética , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Mutagênese/efeitos dos fármacos , Animais , Linhagem Celular , CamundongosRESUMO
In humans, DNA double-strand breaks (DSBs) are repaired by two mutually-exclusive mechanisms, homologous recombination or end-joining. Among end-joining mechanisms, the main process is classical non-homologous end-joining (C-NHEJ) which relies on Ku binding to DNA ends and DNA Ligase IV (Lig4)-mediated ligation. Mostly under Ku- or Lig4-defective conditions, an alternative end-joining process (A-EJ) can operate and exhibits a trend toward microhomology usage at the break junction. Homologous recombination relies on an initial MRN-dependent nucleolytic degradation of one strand at DNA ends. This process, named DNA resection generates 3' single-stranded tails necessary for homologous pairing with the sister chromatid. While it is believed from the current literature that the balance between joining and recombination processes at DSBs ends is mainly dependent on the initiation of resection, it has also been shown that MRN activity can generate short single-stranded DNA oligonucleotides (ssO) that may also be implicated in repair regulation. Here, we evaluate the effect of ssO on end-joining at DSB sites both in vitro and in cells. We report that under both conditions, ssO inhibit C-NHEJ through binding to Ku and favor repair by the Lig4-independent microhomology-mediated A-EJ process.
Assuntos
Antígenos Nucleares/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Células HeLa , Humanos , Autoantígeno Ku , Oligodesoxirribonucleotídeos/metabolismoRESUMO
DNA double-strand breaks (DSBs) are highly cytotoxic lesions that are generated by ionizing radiation and various DNA-damaging chemicals. Following DSB formation, cells activate the DNA-damage response (DDR) protein kinases ATM, ATR and DNA-PK (also known as PRKDC). These then trigger histone H2AX (also known as H2AFX) phosphorylation and the accumulation of proteins such as MDC1, 53BP1 (also known as TP53BP1), BRCA1, CtIP (also known as RBBP8), RNF8 and RNF168/RIDDLIN into ionizing radiation-induced foci (IRIF) that amplify DSB signalling and promote DSB repair. Attachment of small ubiquitin-related modifier (SUMO) to target proteins controls diverse cellular functions. Here, we show that SUMO1, SUMO2 and SUMO3 accumulate at DSB sites in mammalian cells, with SUMO1 and SUMO2/3 accrual requiring the E3 ligase enzymes PIAS4 and PIAS1. We also establish that PIAS1 and PIAS4 are recruited to damage sites via mechanisms requiring their SAP domains, and are needed for the productive association of 53BP1, BRCA1 and RNF168 with such regions. Furthermore, we show that PIAS1 and PIAS4 promote DSB repair and confer ionizing radiation resistance. Finally, we establish that PIAS1 and PIAS4 are required for effective ubiquitin-adduct formation mediated by RNF8, RNF168 and BRCA1 at sites of DNA damage. These findings thus identify PIAS1 and PIAS4 as components of the DDR and reveal how protein recruitment to DSB sites is controlled by coordinated SUMOylation and ubiquitylation.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Animais , Proteína BRCA1/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Biológicos , Fosforilação , Proteínas Inibidoras de STAT Ativados/química , Proteínas Inibidoras de STAT Ativados/genética , Estrutura Terciária de Proteína , Proteína de Replicação A/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
The conserved MRE11RAD50NBS1 (MRN) complex is an important sensor of DNA double-strand breaks (DSBs) and facilitates DNA repair by homologous recombination (HR) and end joining. Here, we identify NBS1 as a target of cyclin-dependent kinase (CDK) phosphorylation. We show that NBS1 serine 432 phosphorylation occurs in the S, G2 and M phases of the cell cycle and requires CDK activity. This modification stimulates MRN-dependent conversion of DSBs into structures that are substrates for repair by HR. Impairment of NBS1 phosphorylation not only negatively affects DSB repair by HR, but also prevents resumption of DNA replication after replication-fork stalling. Thus, CDK-mediated NBS1 phosphorylation defines a molecular switch that controls the choice of repair mode for DSBs.
Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Clivagem do DNA , Replicação do DNA , Recombinação Homóloga , Proteínas Nucleares/metabolismo , Hidrolases Anidrido Ácido , Substituição de Aminoácidos , Proteína Quinase CDC2/química , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Enzimas Reparadoras do DNA/química , Proteínas de Ligação a DNA/química , Humanos , Proteína Homóloga a MRE11 , Mutagênese Sítio-Dirigida , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosforilação , Processamento de Proteína Pós-Traducional , Serina/genética , Serina/metabolismoRESUMO
In the S and G2 phases of the cell cycle, DNA double-strand breaks (DSBs) are processed into single-stranded DNA, triggering ATR-dependent checkpoint signalling and DSB repair by homologous recombination. Previous work has implicated the MRE11 complex in such DSB-processing events. Here, we show that the human CtIP (RBBP8) protein confers resistance to DSB-inducing agents and is recruited to DSBs exclusively in the S and G2 cell-cycle phases. Moreover, we reveal that CtIP is required for DSB resection, and thereby for recruitment of replication protein A (RPA) and the protein kinase ATR to DSBs, and for the ensuing ATR activation. Furthermore, we establish that CtIP physically and functionally interacts with the MRE11 complex, and that both CtIP and MRE11 are required for efficient homologous recombination. Finally, we reveal that CtIP has sequence homology with Sae2, which is involved in MRE11-dependent DSB processing in yeast. These findings establish evolutionarily conserved roles for CtIP-like proteins in controlling DSB resection, checkpoint signalling and homologous recombination.
Assuntos
Proteínas de Transporte/metabolismo , Reparo do DNA , DNA/metabolismo , Proteínas Nucleares/metabolismo , Recombinação Genética , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Sequência Conservada , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases , Endonucleases , Evolução Molecular , Fase G2 , Humanos , Proteína Homóloga a MRE11 , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/metabolismo , Recombinação Genética/efeitos dos fármacos , Fase S , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Ataxia-telangiectasia mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are members of the phosphoinositide-3-kinase-related protein kinase (PIKK) family, and are rapidly activated in response to DNA damage. ATM and DNA-PKcs respond mainly to DNA double-strand breaks, whereas ATR is activated by single-stranded DNA and stalled DNA replication forks. In all cases, activation involves their recruitment to the sites of damage. Here we identify related, conserved carboxy-terminal motifs in human Nbs1, ATRIP and Ku80 proteins that are required for their interaction with ATM, ATR and DNA-PKcs, respectively. These motifs are essential not only for efficient recruitment of ATM, ATR and DNA-PKcs to sites of damage, but are also critical for ATM-, ATR- and DNA-PKcs-mediated signalling events that trigger cell cycle checkpoints and DNA repair. Our findings reveal that recruitment of these PIKKs to DNA lesions occurs by common mechanisms through an evolutionarily conserved motif, and provide direct evidence that PIKK recruitment is required for PIKK-dependent DNA-damage signalling.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Antígenos Nucleares/química , Antígenos Nucleares/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Sequência Conservada , Cricetinae , DNA/genética , DNA/metabolismo , Proteína Quinase Ativada por DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Autoantígeno Ku , Dados de Sequência Molecular , Proteínas Nucleares/genética , Fosfatidilinositol 3-Quinases/química , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Transdução de Sinais , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genéticaRESUMO
The Fanconi anemia pathway orchestrates the repair of DNA interstrand cross-links and stalled replication forks. A key step in this pathway is UBE2T and FANCL-dependent monoubiquitylation of the FANCD2-FANCI complex. The Fanconi anemia pathway represents an attractive therapeutic target, because activation of this pathway has been linked to chemotherapy resistance in several cancers. However, to date, very few selective inhibitors of ubiquitin conjugation pathways are known. By using a high-throughput screen-compatible assay, we have identified a small-molecule inhibitor of UBE2T/FANCL-mediated FANCD2 monoubiquitylation that sensitizes cells to the DNA cross-linking agent, carboplatin.
Assuntos
Proteína do Grupo de Complementação L da Anemia de Fanconi/antagonistas & inibidores , Anemia de Fanconi/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Linhagem Celular Tumoral , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Enzimas de Conjugação de Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Histone H2AX and MDC1 are key DNA repair and DNA-damage signalling proteins. When DNA double-strand breaks (DSBs) occur, H2AX is phosphorylated and then recruits MDC1, which in turn serves as a docking platform to promote the localization of other factors, including 53BP1, to DSB sites. Here, by using CRISPR-Cas9 engineered human cell lines, we identify a hitherto unknown, H2AX-independent, function of MDC1 mediated by its PST-repeat region. We show that the PST-repeat region directly interacts with chromatin via the nucleosome acidic patch and mediates DNA damage-independent association of MDC1 with chromatin. We find that this region is largely functionally dispensable when the canonical γH2AX-MDC1 pathway is operative but becomes critical for 53BP1 recruitment to DNA-damage sites and cell survival following DSB induction when H2AX is not available. Consequently, our results suggest a role for MDC1 in activating the DDR in areas of the genome lacking or depleted of H2AX.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Dano ao DNA , Histonas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Motivos de Aminoácidos , Proteínas de Ciclo Celular/genética , Linhagem Celular , Cromatina/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Histonas/genética , Humanos , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismoRESUMO
Mutations in the ATM tumor suppressor gene confer hypersensitivity to DNA-damaging chemotherapeutic agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase I inhibitor topotecan. Thus, we here establish that inactivating terminal components of the non-homologous end-joining (NHEJ) machinery or of the BRCA1-A complex specifically confer topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase (PARP) inhibitor olaparib reflects delayed engagement of homologous recombination at DNA-replication-fork associated single-ended double-strand breaks (DSBs), allowing some to be subject to toxic NHEJ. Preventing DSB ligation by NHEJ, or enhancing homologous recombination by BRCA1-A complex disruption, suppresses this toxicity, highlighting a crucial role for ATM in preventing toxic LIG4-mediated chromosome fusions. Notably, suppressor mutations in ATM-mutant backgrounds are different to those in BRCA1-mutant scenarios, suggesting new opportunities for patient stratification and additional therapeutic vulnerabilities for clinical exploitation.
Assuntos
Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Reparo do DNA por Junção de Extremidades/genética , Resistencia a Medicamentos Antineoplásicos/genética , Animais , Antineoplásicos/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Ligase Dependente de ATP/metabolismo , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Células-Tronco Embrionárias Murinas , Mutação , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Ftalazinas/farmacologia , Ftalazinas/uso terapêutico , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Topotecan/farmacologia , Topotecan/uso terapêuticoRESUMO
Establishing genetic and chemo-genetic interactions has played key roles in elucidating mechanisms by which certain chemicals perturb cellular functions. In contrast to gene disruption/depletion strategies to identify mechanisms of drug resistance, searching for point-mutational genetic suppressors that can identify separation- or gain-of-function mutations has been limited. Here, by demonstrating its utility in identifying chemical-genetic suppressors of sensitivity to the DNA topoisomerase I poison camptothecin or the poly(ADP-ribose) polymerase inhibitor olaparib, we detail an approach allowing systematic, large-scale detection of spontaneous or chemically-induced suppressor mutations in yeast or haploid mammalian cells in a short timeframe, and with potential applications in other haploid systems. In addition to applications in molecular biology research, this protocol can be used to identify drug targets and predict drug-resistance mechanisms. Mapping suppressor mutations on the primary or tertiary structures of protein suppressor hits provides insights into functionally relevant protein domains. Importantly, we show that olaparib resistance is linked to missense mutations in the DNA binding regions of PARP1, but not in its catalytic domain. This provides experimental support to the concept of PARP1 trapping on DNA as the prime source of toxicity to PARP inhibitors, and points to a novel olaparib resistance mechanism with potential therapeutic implications.
Assuntos
Camptotecina/farmacologia , DNA Topoisomerases Tipo I/genética , Testes Genéticos , Estudo de Associação Genômica Ampla , Domínios Proteicos/genética , Domínios e Motivos de Interação entre Proteínas , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Células-Tronco Embrionárias , Humanos , Camundongos , Modelos Moleculares , Mutação , Poli(ADP-Ribose) Polimerase-1/química , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Conformação ProteicaRESUMO
BRCA1 deficiencies cause breast, ovarian, prostate and other cancers, and render tumours hypersensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. To understand the resistance mechanisms, we conducted whole-genome CRISPR-Cas9 synthetic-viability/resistance screens in BRCA1-deficient breast cancer cells treated with PARP inhibitors. We identified two previously uncharacterized proteins, C20orf196 and FAM35A, whose inactivation confers strong PARP-inhibitor resistance. Mechanistically, we show that C20orf196 and FAM35A form a complex, 'Shieldin' (SHLD1/2), with FAM35A interacting with single-stranded DNA through its C-terminal oligonucleotide/oligosaccharide-binding fold region. We establish that Shieldin acts as the downstream effector of 53BP1/RIF1/MAD2L2 to promote DNA double-strand break (DSB) end-joining by restricting DSB resection and to counteract homologous recombination by antagonizing BRCA2/RAD51 loading in BRCA1-deficient cells. Notably, Shieldin inactivation further sensitizes BRCA1-deficient cells to cisplatin, suggesting how defining the SHLD1/2 status of BRCA1-deficient tumours might aid patient stratification and yield new treatment opportunities. Highlighting this potential, we document reduced SHLD1/2 expression in human breast cancers displaying intrinsic or acquired PARP-inhibitor resistance.
Assuntos
Proteína BRCA1/genética , Neoplasias Ósseas/tratamento farmacológico , Neoplasias da Mama/tratamento farmacológico , Reparo do DNA por Junção de Extremidades , Resistencia a Medicamentos Antineoplásicos , Osteossarcoma/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas/metabolismo , Reparo de DNA por Recombinação , Animais , Proteína BRCA1/deficiência , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Cisplatino/farmacologia , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Células HEK293 , Humanos , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Camundongos , Complexos Multiproteicos , Osteossarcoma/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteínas/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
This corrects the article DOI: 10.1038/ncomms12889.
RESUMO
Repair of single-ended DNA double-strand breaks (seDSBs) by homologous recombination (HR) requires the generation of a 3' single-strand DNA overhang by exonuclease activities in a process called DNA resection. However, it is anticipated that the highly abundant DNA end-binding protein Ku sequesters seDSBs and shields them from exonuclease activities. Despite pioneering works in yeast, it is unclear how mammalian cells counteract Ku at seDSBs to allow HR to proceed. Here we show that in human cells, ATM-dependent phosphorylation of CtIP and the epistatic and coordinated actions of MRE11 and CtIP nuclease activities are required to limit the stable loading of Ku on seDSBs. We also provide evidence for a hitherto unsuspected additional mechanism that contributes to prevent Ku accumulation at seDSBs, acting downstream of MRE11 endonuclease activity and in parallel with MRE11 exonuclease activity. Finally, we show that Ku persistence at seDSBs compromises Rad51 focus assembly but not DNA resection.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Transporte/metabolismo , Quebras de DNA de Cadeia Dupla , Autoantígeno Ku/metabolismo , Proteína Homóloga a MRE11/metabolismo , Proteínas Nucleares/metabolismo , Linhagem Celular Tumoral , Reparo do DNA , Endodesoxirribonucleases , Humanos , Fosforilação , Rad51 Recombinase/metabolismoRESUMO
The BRCA1 tumor suppressor plays an important role in homologous recombination (HR)-mediated DNA double-strand-break (DSB) repair. BRCA1 is phosphorylated by Chk2 kinase upon γ-irradiation, but the role of Chk2 phosphorylation is not understood. Here, we report that abrogation of Chk2 phosphorylation on BRCA1 delays end resection and the dispersion of BRCA1 from DSBs but does not affect the assembly of Mre11/Rad50/NBS1 (MRN) and CtIP at DSBs. Moreover, we show that BRCA1 is ubiquitinated by SCF(Skp2) and that abrogation of Chk2 phosphorylation impairs its ubiquitination. Our study suggests that BRCA1 is more than a scaffold protein to assemble HR repair proteins at DSBs, but that Chk2 phosphorylation of BRCA1 also serves as a built-in clock for HR repair of DSBs. BRCA1 is known to inhibit Mre11 nuclease activity. SCF(Skp2) activity appears at late G1 and peaks at S/G2, and is known to ubiquitinate phosphodegron motifs. The removal of BRCA1 from DSBs by SCF(Skp2)-mediated degradation terminates BRCA1-mediated inhibition of Mre11 nuclease activity, allowing for end resection and restricting the initiation of HR to the S/G2 phases of the cell cycle.
Assuntos
Proteína BRCA1/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Dano ao DNA , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HEK293 , Humanos , Camundongos , Complexos Multiproteicos/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Estabilidade Proteica/efeitos dos fármacos , Proteína de Replicação A/metabolismo , Fatores de Tempo , Ubiquitinação/efeitos dos fármacosRESUMO
Ubiquitylation is crucial for proper cellular responses to DNA double-strand breaks (DSBs). If unrepaired, these highly cytotoxic lesions cause genome instability, tumorigenesis, neurodegeneration or premature ageing. Here, we conduct a comprehensive, multilayered screen to systematically profile all human ubiquitin E2 enzymes for impacts on cellular DSB responses. With a widely applicable approach, we use an exemplary E2 family, UBE2Ds, to identify ubiquitylation-cascade components downstream of E2s. Thus, we uncover the nuclear E3 ligase RNF138 as a key homologous recombination (HR)-promoting factor that functions with UBE2Ds in cells. Mechanistically, UBE2Ds and RNF138 accumulate at DNA-damage sites and act at early resection stages by promoting CtIP ubiquitylation and accrual. This work supplies insights into regulation of DSB repair by HR. Moreover, it provides a rich information resource on E2s that can be exploited by follow-on studies.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Nucleares/metabolismo , Reparo de DNA por Recombinação , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Transporte/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Endodesoxirribonucleases , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Immunoblotting , Microscopia Confocal , Proteínas Nucleares/genética , Interferência de RNA , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
XRCC4 and XLF are two structurally related proteins that function in DNA double-strand break (DSB) repair. Here, we identify human PAXX (PAralog of XRCC4 and XLF, also called C9orf142) as a new XRCC4 superfamily member and show that its crystal structure resembles that of XRCC4. PAXX interacts directly with the DSB-repair protein Ku and is recruited to DNA-damage sites in cells. Using RNA interference and CRISPR-Cas9 to generate PAXX(-/-) cells, we demonstrate that PAXX functions with XRCC4 and XLF to mediate DSB repair and cell survival in response to DSB-inducing agents. Finally, we reveal that PAXX promotes Ku-dependent DNA ligation in vitro and assembly of core nonhomologous end-joining (NHEJ) factors on damaged chromatin in cells. These findings identify PAXX as a new component of the NHEJ machinery.
Assuntos
Antígenos Nucleares/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/metabolismo , Linhagem Celular Tumoral , Cristalografia por Raios X , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Autoantígeno Ku , Estrutura Secundária de Proteína , Interferência de RNARESUMO
Mammalian CtIP protein has major roles in DNA double-strand break (DSB) repair. Although it is well established that CtIP promotes DNA-end resection in preparation for homology-dependent DSB repair, the molecular basis for this function has remained unknown. Here we show by biophysical and X-ray crystallographic analyses that the N-terminal domain of human CtIP exists as a stable homotetramer. Tetramerization results from interlocking interactions between the N-terminal extensions of CtIP's coiled-coil region, which lead to a 'dimer-of-dimers' architecture. Through interrogation of the CtIP structure, we identify a point mutation that abolishes tetramerization of the N-terminal domain while preserving dimerization in vitro. Notably, we establish that this mutation abrogates CtIP oligomer assembly in cells, thus leading to strong defects in DNA-end resection and gene conversion. These findings indicate that the CtIP tetramer architecture described here is essential for effective DSB repair by homologous recombination.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Multimerização Proteica/fisiologia , Cristalografia por Raios X , Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Endodesoxirribonucleases , HumanosRESUMO
DNA double-strand breaks (DSBs) are the most toxic of all genomic insults, and pathways dealing with their signaling and repair are crucial to prevent cancer and for immune system development. Despite intense investigations, our knowledge of these pathways has been technically limited by our inability to detect the main repair factors at DSBs in cells. In this paper, we present an original method that involves a combination of ribonuclease- and detergent-based preextraction with high-resolution microscopy. This method allows direct visualization of previously hidden repair complexes, including the main DSB sensor Ku, at virtually any type of DSB, including those induced by anticancer agents. We demonstrate its broad range of applications by coupling it to laser microirradiation, super-resolution microscopy, and single-molecule counting to investigate the spatial organization and composition of repair factories. Furthermore, we use our method to monitor DNA repair and identify mechanisms of repair pathway choice, and we show its utility in defining cellular sensitivities and resistance mechanisms to anticancer agents.
Assuntos
Antígenos Nucleares/química , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/química , Microscopia/métodos , Animais , Antígenos Nucleares/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Humanos , Autoantígeno Ku , CamundongosRESUMO
Chromosomal deletions and rearrangements in tumors are often associated with common fragile sites, which are specific genomic loci prone to gaps and breaks in metaphase chromosomes. Common fragile sites appear to arise through incomplete DNA replication because they are induced after partial replication inhibition by agents such as aphidicolin. Here, we show that in G1 cells, large nuclear bodies arise that contain p53 binding protein 1 (53BP1), phosphorylated H2AX (γH2AX), and mediator of DNA damage checkpoint 1 (MDC1), as well as components of previously characterized OPT (Oct-1, PTF, transcription) domains. Notably, we find that incubating cells with low aphidicolin doses increases the incidence and number of 53BP1-OPT domains in G1 cells, and by chromatin immunoprecipitation and massively parallel sequencing analysis of γH2AX, we demonstrate that OPT domains are enriched at common fragile sites. These findings invoke a model wherein incomplete DNA synthesis during S phase leads to a DNA damage response and formation of 53BP1-OPT domains in the subsequent G1.