RESUMO
When replication forks encounter damaged DNA, cells utilize damage tolerance mechanisms to allow replication to proceed. These include translesion synthesis at the fork, postreplication gap filling, and template switching via fork reversal or homologous recombination. The extent to which these different damage tolerance mechanisms are utilized depends on cell, tissue, and developmental context-specific cues, the last two of which are poorly understood. To address this gap, we have investigated damage tolerance responses in Drosophila melanogaster. We report that tolerance of DNA alkylation damage in rapidly dividing larval tissues depends heavily on translesion synthesis. Furthermore, we show that the REV1 protein plays a multi-faceted role in damage tolerance in Drosophila. Larvae lacking REV1 are hypersensitive to methyl methanesulfonate (MMS) and have highly elevated levels of γ-H2Av (Drosophila γ-H2AX) foci and chromosome aberrations in MMS-treated tissues. Loss of the REV1 C-terminal domain (CTD), which recruits multiple translesion polymerases to damage sites, sensitizes flies to MMS. In the absence of the REV1 CTD, DNA polymerases eta and zeta become critical for MMS tolerance. In addition, flies lacking REV3, the catalytic subunit of polymerase zeta, require the deoxycytidyl transferase activity of REV1 to tolerate MMS. Together, our results demonstrate that Drosophila prioritize the use of multiple translesion polymerases to tolerate alkylation damage and highlight the critical role of REV1 in the coordination of this response to prevent genome instability.
Assuntos
Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA , Proteínas de Drosophila , Drosophila melanogaster , Metanossulfonato de Metila , Nucleotidiltransferases , Animais , Drosophila melanogaster/genética , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Metanossulfonato de Metila/farmacologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Alquilação , Reparo do DNA/genética , Replicação do DNA/genética , Larva/genética , Histonas/metabolismo , Histonas/genéticaRESUMO
When replication forks encounter damaged DNA, cells utilize DNA damage tolerance mechanisms to allow replication to proceed. These include translesion synthesis at the fork, postreplication gap filling, and template switching via fork reversal or homologous recombination. The extent to which these different damage tolerance mechanisms are utilized depends on cell, tissue, and developmental context-specific cues, the last two of which are poorly understood. To address this gap, we have investigated damage tolerance responses following alkylation damage in Drosophila melanogaster. We report that translesion synthesis, rather than template switching, is the preferred response to alkylation-induced damage in diploid larval tissues. Furthermore, we show that the REV1 protein plays a multi-faceted role in damage tolerance in Drosophila. Drosophila larvae lacking REV1 are hypersensitive to methyl methanesulfonate (MMS) and have highly elevated levels of γ-H2Av foci and chromosome aberrations in MMS-treated tissues. Loss of the REV1 C-terminal domain (CTD), which recruits multiple translesion polymerases to damage sites, sensitizes flies to MMS. In the absence of the REV1 CTD, DNA polymerases eta and zeta become critical for MMS tolerance. In addition, flies lacking REV3, the catalytic subunit of polymerase zeta, require the deoxycytidyl transferase activity of REV1 to tolerate MMS. Together, our results demonstrate that Drosophila prioritize the use of multiple translesion polymerases to tolerate alkylation damage and highlight the critical role of REV1 in the coordination of this response to prevent genome instability.
RESUMO
The 53BP1-RIF1 pathway restricts the resection of DNA double-strand breaks (DSBs) and promotes blunt end-ligation by non-homologous end joining (NHEJ) repair. The Shieldin complex is a downstream effector of the 53BP1-RIF1 pathway. Here, we identify a component of this pathway, CCAR2/DBC1, which is also required for restriction of DNA end-resection. CCAR2 co-immunoprecipitates with the Shieldin complex, and knockout of CCAR2 in a BRCA1-deficient cell line results in elevated DSB end-resection, RAD51 loading, and PARP inhibitor (PARPi) resistance. Knockout of CCAR2 is epistatic with knockout of other Shieldin proteins. The S1-like RNA-binding domain of CCAR2 is required for its interaction with the Shieldin complex and for suppression of DSB end-resection. CCAR2 functions downstream of the Shieldin complex, and CCAR2 knockout cells have delayed resolution of Shieldin complex foci. Forkhead-associated (FHA)-dependent targeting of CCAR2 to DSB sites re-sensitized BRCA1-/-SHLD2-/- cells to PARPi. Taken together, CCAR2 is a functional component of the 53BP1-RIF1 pathway, promotes the refill of resected DSBs, and suppresses homologous recombination.
Assuntos
Quebras de DNA de Cadeia Dupla , Inibidores de Poli(ADP-Ribose) Polimerases , Reparo do DNA por Junção de Extremidades , Recombinação Homóloga , DNARESUMO
A critical determinant of DNA repair pathway choice is REV7, an adaptor that binds to various DNA repair proteins through its C-terminal seatbelt domain. The REV7 seatbelt binds to either REV3, activating translesion synthesis, or to SHLD3, activating non-homologous end joining (NHEJ) repair. Recent studies have identified another REV7 seatbelt-binding protein, CHAMP1 (chromosome alignment-maintaining phosphoprotein 1), though its possible role in DNA repair is unknown. Here, we show that binding of CHAMP1 to REV7 activates homologous recombination (HR) repair. Mechanistically, CHAMP1 binds directly to REV7 and reduces the level of the Shieldin complex, causing an increase in double-strand break end resection. CHAMP1 also interacts with POGZ in a heterochromatin complex further promoting HR repair. Importantly, in human tumors, CHAMP1 overexpression promotes HR, confers poly (ADP-ribose) polymerase inhibitor resistance, and correlates with poor prognosis. Thus, by binding to either SHLD3 or CHAMP1 through its seatbelt, the REV7 protein can promote either NHEJ or HR repair, respectively.
Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Proteínas Mad2 , Reparo de DNA por Recombinação , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Reparo do DNA por Junção de Extremidades , Reparo do DNA/genética , Recombinação Homóloga , Humanos , Proteínas Mad2/metabolismo , Fosfoproteínas/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Reparo de DNA por Recombinação/genética , Transposases/metabolismoRESUMO
Protection of stalled replication forks is critical to genomic stability. Using genetic and proteomic analyses, we discovered the Protexin complex containing the ssDNA binding protein SCAI and the DNA polymerase REV3. Protexin is required specifically for protecting forks stalled by nucleotide depletion, fork barriers, fragile sites, and DNA inter-strand crosslinks (ICLs), where it promotes homologous recombination and repair. Protexin loss leads to ssDNA accumulation and profound genomic instability in response to ICLs. Protexin interacts with RNA POL2, and both oppose EXO1's resection of DNA on forks remodeled by the FANCM translocase activity. This pathway acts independently of BRCA/RAD51-mediated fork stabilization, and cells with BRCA2 mutations were dependent on SCAI for survival. These data suggest that Protexin and its associated factors establish a new fork protection pathway that counteracts fork resection in part through a REV3 polymerase-dependent resynthesis mechanism of excised DNA, particularly at ICL stalled forks.
Assuntos
Proteína BRCA2/metabolismo , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/química , DNA Polimerase Dirigida por DNA/química , Exodesoxirribonucleases/metabolismo , Fatores de Transcrição/química , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Reparo do DNA , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Células HeLa , Humanos , Ácido Mevalônico , Camundongos , Complexos Multiproteicos , Mutação , Ligação Proteica , Conformação Proteica , RNA Guia de Cinetoplastídeos/metabolismo , RNA Interferente Pequeno/metabolismo , Recombinação GenéticaRESUMO
REV7 is a small multifunctional protein that participates in multiple DNA repair pathways, most notably translesion DNA synthesis and double-strand break (DSB) repair. While the role of REV7 in translesion synthesis has been known for several decades, its function in DSB repair is a recent discovery. Investigations into the DSB repair function of REV7 have led to the discovery of a new DNA repair complex known as Shieldin. Recent studies have also highlighted the importance of REV7's HORMA domain, an ancient structural motif, in REV7 function and have identified the HORMA regulators, TRIP13 and p31, as novel DNA repair factors. In this review, we discuss these recent findings and their implications for repair pathway choice, at both DSBs and replication forks. We suggest that REV7, in particular the activation state of its HORMA domain, can act as a critical determinant of mutagenic versus error-free repair in multiple contexts.
Assuntos
Proteínas de Ciclo Celular , Reparo do DNA , Proteínas Mad2 , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Proteínas Mad2/genética , Proteínas Mad2/metabolismoRESUMO
Homologous recombination (HR)-deficient cancers are sensitive to poly-ADP ribose polymerase inhibitors (PARPi), which have shown clinical efficacy in the treatment of high-grade serous cancers (HGSC). However, the majority of patients will relapse, and acquired PARPi resistance is emerging as a pressing clinical problem. Here we generated seven single-cell clones with acquired PARPi resistance derived from a PARPi-sensitive TP53 -/- and BRCA1 -/- epithelial cell line generated using CRISPR/Cas9. These clones showed diverse resistance mechanisms, and some clones presented with multiple mechanisms of resistance at the same time. Genomic analysis of the clones revealed unique transcriptional and mutational profiles and increased genomic instability in comparison with a PARPi-sensitive cell line. Clonal evolutionary analyses suggested that acquired PARPi resistance arose via clonal selection from an intrinsically unstable and heterogenous cell population in the sensitive cell line, which contained preexisting drug-tolerant cells. Similarly, clonal and spatial heterogeneity in tumor biopsies from a clinical patient with BRCA1-mutant HGSC with acquired PARPi resistance was observed. In an imaging-based drug screening, the clones showed heterogenous responses to targeted therapeutic agents, indicating that not all PARPi-resistant clones can be targeted with just one therapy. Furthermore, PARPi-resistant clones showed mechanism-dependent vulnerabilities to the selected agents, demonstrating that a deeper understanding on the mechanisms of resistance could lead to improved targeting and biomarkers for HGSC with acquired PARPi resistance. SIGNIFICANCE: This study shows that BRCA1-deficient cells can give rise to multiple genomically and functionally heterogenous PARPi-resistant clones, which are associated with various vulnerabilities that can be targeted in a mechanism-specific manner.
Assuntos
Proteína BRCA1/fisiologia , Evolução Clonal , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Apoptose , Proliferação de Células , Feminino , Instabilidade Genômica , Recombinação Homóloga , Humanos , Camundongos , Camundongos Knockout , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Transcriptoma , Células Tumorais CultivadasRESUMO
The repair of DNA double strand breaks (DSBs) that arise from external mutagenic agents and routine cellular processes is essential for life. DSBs are repaired by two major pathways, homologous recombination (HR) and classical nonhomologous end joining (C-NHEJ). DSB repair pathway choice is largely dictated at the step of 5'-3' DNA end resection, which is promoted during S phase, in part by BRCA1. Opposing end resection is the 53BP1 protein, which recruits the ssDNA-binding REV7-Shieldin complex to favor C-NHEJ repair. We recently identified TRIP13 as a proresection factor that remodels REV7, causing its dissociation from the Shieldin subunit SHLD3. Here, we identify p31comet, a negative regulator of MAD2 and the spindle assembly checkpoint, as an important mediator of the TRIP13-REV7 interaction. p31comet binds to the REV7-Shieldin complex in cells, promotes REV7 inactivation, and causes PARP inhibitor resistance. p31comet also participates in the extraction of REV7 from the chromatin. Furthermore, p31comet can counteract REV7 function in translesion synthesis (TLS) by releasing it from REV3 in the Pol ζ complex. Finally, p31comet, like TRIP13, is overexpressed in many cancers and this correlates with poor prognosis. Thus, we reveal a key player in the regulation of HR and TLS with significant clinical implications.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Mad2/metabolismo , Proteínas Nucleares/metabolismo , Reparo de DNA por Recombinação , Linhagem Celular Tumoral , Células HEK293 , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/mortalidadeRESUMO
In the past decade, the study of the major DNA double strand break (DSB) repair pathways, homologous recombination (HR) and classical non-homologous end joining (C-NHEJ), has revealed a vast and intricate network of regulation. The choice between HR and C-NHEJ is largely controlled at the step of DNA end-resection. A pro-C-NHEJ cascade commencing with 53BP1 and culminating in the newly discovered REV7-Shieldin complex impedes end resection and therefore HR. Importantly, loss of any component of this pathway confers PARP inhibitor resistance in BRCA1-deficient cells; hence, their study is of great clinical importance. The newest entrant on the scene of end resection regulation is the ATPase TRIP13 that disables the pro-C-NHEJ cascade by promoting a novel conformational change of the HORMA protein REV7. Here, we tie these new findings and factors with previous research on the regulation of DSB repair and HORMA proteins, and suggest testable hypotheses for how TRIP13 could specifically inactivate REV7-Shieldin to promote HR. We also discuss these biological questions in the context of clinical therapeutics.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Complexos Multiproteicos/metabolismo , Sequência de Aminoácidos , Animais , Instabilidade Genômica , Humanos , Modelos Moleculares , Complexos Multiproteicos/química , Ligação ProteicaRESUMO
DNA double-strand breaks (DSBs) are repaired through homology-directed repair (HDR) or non-homologous end joining (NHEJ). BRCA1/2-deficient cancer cells cannot perform HDR, conferring sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPi). However, concomitant loss of the pro-NHEJ factors 53BP1, RIF1, REV7-Shieldin (SHLD1-3) or CST-DNA polymerase alpha (Pol-α) in BRCA1-deficient cells restores HDR and PARPi resistance. Here, we identify the TRIP13 ATPase as a negative regulator of REV7. We show that REV7 exists in active 'closed' and inactive 'open' conformations, and TRIP13 catalyses the inactivating conformational change, thereby dissociating REV7-Shieldin to promote HDR. TRIP13 similarly disassembles the REV7-REV3 translesion synthesis (TLS) complex, a component of the Fanconi anaemia pathway, inhibiting error-prone replicative lesion bypass and interstrand crosslink repair. Importantly, TRIP13 overexpression is common in BRCA1-deficient cancers, confers PARPi resistance and correlates with poor prognosis. Thus, TRIP13 emerges as an important regulator of DNA repair pathway choice-promoting HDR, while suppressing NHEJ and TLS.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Proteína BRCA1/deficiência , Proteínas de Ciclo Celular/genética , Reparo do DNA/genética , Reparo de DNA por Recombinação/genética , ATPases Associadas a Diversas Atividades Celulares/efeitos dos fármacos , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Humanos , Proteínas Mad2/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas de Ligação a Telômeros/efeitos dos fármacos , Proteínas de Ligação a Telômeros/genéticaRESUMO
BRCA1 or BRCA2 inactivation drives breast and ovarian cancer but also creates vulnerability to poly(ADP-ribose) polymerase (PARP) inhibitors. To search for additional targets whose inhibition is synthetically lethal in BRCA2-deficient backgrounds, we screened two pairs of BRCA2 isogenic cell lines with DNA-repair-focused small hairpin RNA (shRNA) and CRISPR (clustered regularly interspaced short palindromic repeats)-based libraries. We found that BRCA2-deficient cells are selectively dependent on multiple pathways including base excision repair, ATR signaling, and splicing. We identified APEX2 and FEN1 as synthetic lethal genes with both BRCA1 and BRCA2 loss of function. BRCA2-deficient cells require the apurinic endonuclease activity and the PCNA-binding domain of Ape2 (APEX2), but not Ape1 (APEX1). Furthermore, BRCA2-deficient cells require the 5' flap endonuclease but not the 5'-3' exonuclease activity of Fen1, and chemically inhibiting Fen1 selectively targets BRCA-deficient cells. Finally, we developed a microhomology-mediated end-joining (MMEJ) reporter and showed that Fen1 participates in MMEJ, underscoring the importance of MMEJ as a collateral repair pathway in the context of homologous recombination (HR) deficiency.
Assuntos
Proteína BRCA2/genética , Sistemas CRISPR-Cas , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Endonucleases Flap/genética , Genes Letais , Neoplasias/genética , Interferência de RNA , Mutações Sintéticas Letais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Morte Celular , Linhagem Celular Tumoral , Dano ao DNA , Reparo do DNA por Junção de Extremidades , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endonucleases , Endonucleases Flap/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Enzimas Multifuncionais , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Interferente Pequeno/genéticaRESUMO
BRCA1-deficient tumor cells have defects in homologous-recombination repair and replication fork stability, resulting in PARP inhibitor sensitivity. Here, we demonstrate that a deubiquitinase, USP1, is upregulated in tumors with mutations in BRCA1. Knockdown or inhibition of USP1 resulted in replication fork destabilization and decreased viability of BRCA1-deficient cells, revealing a synthetic lethal relationship. USP1 binds to and is stimulated by fork DNA. A truncated form of USP1, lacking its DNA-binding region, was not stimulated by DNA and failed to localize and protect replication forks. Persistence of monoubiquitinated PCNA at the replication fork was the mechanism of cell death in the absence of USP1. Taken together, USP1 exhibits DNA-mediated activation at the replication fork, protects the fork, and promotes survival in BRCA1-deficient cells. Inhibition of USP1 may be a useful treatment for a subset of PARP-inhibitor-resistant BRCA1-deficient tumors with acquired replication fork stabilization.
Assuntos
Proteína BRCA1/deficiência , Neoplasias da Mama/enzimologia , Replicação do DNA , DNA de Neoplasias/biossíntese , Proteases Específicas de Ubiquitina/metabolismo , Neoplasias do Colo do Útero/enzimologia , Animais , Proteína BRCA1/genética , Sítios de Ligação , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células , Sobrevivência Celular , DNA de Neoplasias/genética , Resistência a Medicamentos , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Camundongos Nus , Mutação , Desnaturação de Ácido Nucleico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Proteases Específicas de Ubiquitina/antagonistas & inibidores , Proteases Específicas de Ubiquitina/genética , Ubiquitinação , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Fanconi anemia (FA) is a recessive genetic disease characterized by congenital abnormalities, chromosome instability, progressive bone marrow failure (BMF), and a strong predisposition to cancer. Twenty FA genes have been identified, and the FANC proteins they encode cooperate in a common pathway that regulates DNA crosslink repair and replication fork stability. We identified a child with severe BMF who harbored biallelic inactivating mutations of the translesion DNA synthesis (TLS) gene REV7 (also known as MAD2L2), which encodes the mutant REV7 protein REV7-V85E. Patient-derived cells demonstrated an extended FA phenotype, which included increased chromosome breaks and G2/M accumulation upon exposure to DNA crosslinking agents, γH2AX and 53BP1 foci accumulation, and enhanced p53/p21 activation relative to cells derived from healthy patients. Expression of WT REV7 restored normal cellular and functional phenotypes in the patient's cells, and CRISPR/Cas9 inactivation of REV7 in a non-FA human cell line produced an FA phenotype. Finally, silencing Rev7 in primary hematopoietic cells impaired progenitor function, suggesting that the DNA repair defect underlies the development of BMF in FA. Taken together, our genetic and functional analyses identified REV7 as a previously undescribed FA gene, which we term FANCV.
Assuntos
Anemia de Fanconi/genética , Proteínas Mad2/genética , Mutação , Alelos , Animais , Ciclo Celular , Linhagem Celular Tumoral , Criança , Instabilidade Cromossômica , Quebra Cromossômica , Estudos de Coortes , Reagentes de Ligações Cruzadas/química , Dano ao DNA , Reparo do DNA , Feminino , Fibroblastos/metabolismo , Inativação Gênica , Teste de Complementação Genética , Predisposição Genética para Doença , Variação Genética , Células-Tronco Hematopoéticas/citologia , Humanos , Lentivirus , Proteínas Mad2/metabolismo , Masculino , Camundongos , Camundongos Knockout , Mitose , FenótipoRESUMO
DNA is susceptible to damage by a wide variety of chemical agents that are generated either as byproducts of cellular metabolism or exposure to man-made and harmful environments. Therefore, to maintain genomic integrity, having reliable DNA repair systems is important. DNA polymerase ß is known to be a key player in the base excision repair pathway, and mice devoid of DNA polymerase beta do not live beyond a few hours after birth. In this study, we characterized mice harboring an impaired pol ß variant. This Y265C pol ß variant exhibits slow DNA polymerase activity but WT lyase activity and has been shown to be a mutator polymerase. Mice expressing Y265C pol ß are born at normal Mendelian ratios. However, they are small, and 60% die within a few hours after birth. Slow proliferation and significantly increased levels of cell death are observed in many organs of the E14 homozygous embryos compared with WT littermates. Mouse embryo fibroblasts prepared from the Y265C pol ß embryos proliferate at a rate slower than WT cells and exhibit a gap-filling deficiency during base excision repair. As a result of this, chromosomal aberrations and single- and double-strand breaks are present at significantly higher levels in the homozygous mutant versus WT mouse embryo fibroblasts. This is study in mice is unique in that two enzymatic activities of pol ß have been separated; the data clearly demonstrate that the DNA polymerase activity of pol ß is essential for survival and genome stability.