RESUMO
A cell's ability to survive and to evade cancer is contingent on its ability to retain genomic integrity, which can be seriously compromised when nucleic acid phosphodiester bonds are disrupted. DNA Ligase 1 (LIG1) plays a key role in genome maintenance by sealing single-stranded nicks that are produced during DNA replication and repair. Autosomal recessive mutations in a limited number of individuals have been previously described for this gene. Here we report a homozygous LIG1 mutation (p.A624T), affecting a universally conserved residue, in a patient presenting with leukopenia, neutropenia, lymphopenia, pan-hypogammaglobulinemia, and diminished in vitro response to mitogen stimulation. Patient fibroblasts expressed normal levels of LIG1 protein but exhibited impaired growth, poor viability, high baseline levels of gamma-H2AX foci, and an enhanced susceptibility to DNA-damaging agents. The mutation reduced LIG1 activity by lowering its affinity for magnesium 2.5-fold. Remarkably, it also increased LIG1 fidelity > 50-fold against 3' end 8-Oxoguanine mismatches, exhibiting a marked reduction in its ability to process such nicks. This is expected to yield increased ss- and dsDNA breaks. Molecular dynamic simulations, and Residue Interaction Network studies, predicted an allosteric effect for this mutation on the protein loops associated with the LIG1 high-fidelity magnesium, as well as on DNA binding within the adenylation domain. These dual alterations of suppressed activity and enhanced fidelity, arising from a single mutation, underscore the mechanistic picture of how a LIG1 defect can lead to severe immunological disease.
Assuntos
DNA Ligase Dependente de ATP , Homozigoto , Mutação , Imunodeficiência Combinada Severa , Feminino , Humanos , Masculino , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/metabolismo , Fibroblastos , Simulação de Dinâmica Molecular , Mutação/genética , Imunodeficiência Combinada Severa/genética , LactenteRESUMO
Cyclosporin A (CsA) induces DNA double-strand breaks in LIG4 syndrome fibroblasts, specifically upon transit through S-phase. The basis underlying this has not been described. CsA-induced genomic instability may reflect a direct role of Cyclophilin A (CYPA) in DNA repair. CYPA is a peptidyl-prolyl cis-trans isomerase (PPI). CsA inhibits the PPI activity of CYPA. Using an integrated approach involving CRISPR/Cas9-engineering, siRNA, BioID, co-immunoprecipitation, pathway-specific DNA repair investigations as well as protein expression interaction analysis, we describe novel impacts of CYPA loss and inhibition on DNA repair. We characterise a direct CYPA interaction with the NBS1 component of the MRE11-RAD50-NBS1 complex, providing evidence that CYPA influences DNA repair at the level of DNA end resection. We define a set of genetic vulnerabilities associated with CYPA loss and inhibition, identifying DNA replication fork protection as an important determinant of viability. We explore examples of how CYPA inhibition may be exploited to selectively kill cancers sharing characteristic genomic instability profiles, including MYCN-driven Neuroblastoma, Multiple Myeloma and Chronic Myelogenous Leukaemia. These findings propose a repurposing strategy for Cyclophilin inhibitors.
Assuntos
Hidrolases Anidrido Ácido , Proteínas de Ciclo Celular , Ciclofilina A , Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA , Proteína Homóloga a MRE11 , Proteínas Nucleares , Humanos , Proteína Homóloga a MRE11/metabolismo , Proteína Homóloga a MRE11/genética , Ciclofilina A/metabolismo , Ciclofilina A/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Hidrolases Anidrido Ácido/metabolismo , Hidrolases Anidrido Ácido/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Quebras de DNA de Cadeia Dupla , DNA Ligase Dependente de ATP/metabolismo , DNA Ligase Dependente de ATP/genética , Instabilidade GenômicaRESUMO
TIMELESS (TIM) in the fork protection complex acts as a scaffold of the replisome to prevent its uncoupling and ensure efficient DNA replication fork progression. Nevertheless, its underlying basis for coordinating leading and lagging strand synthesis to limit single-stranded DNA (ssDNA) exposure remains elusive. Here, we demonstrate that acute degradation of TIM at ongoing DNA replication forks induces the accumulation of ssDNA gaps stemming from defective Okazaki fragment (OF) processing. Cells devoid of TIM fail to support the poly(ADP-ribosyl)ation necessary for backing up the canonical OF processing mechanism mediated by LIG1 and FEN1. Consequently, recruitment of XRCC1, a known effector of PARP1-dependent single-strand break repair, to post-replicative ssDNA gaps behind replication forks is impaired. Physical disruption of the TIM-PARP1 complex phenocopies the rapid loss of TIM, indicating that the TIM-PARP1 interaction is critical for the activation of this compensatory pathway. Accordingly, combined deficiency of FEN1 and the TIM-PARP1 interaction leads to synergistic DNA damage and cytotoxicity. We propose that TIM is essential for the engagement of PARP1 to the replisome to coordinate lagging strand synthesis with replication fork progression. Our study identifies TIM as a synthetic lethal target of OF processing enzymes that can be exploited for cancer therapy.
Assuntos
Proteínas de Ciclo Celular , Replicação do DNA , DNA de Cadeia Simples , Peptídeos e Proteínas de Sinalização Intracelular , Poli(ADP-Ribose) Polimerase-1 , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , DNA/metabolismo , DNA/genética , DNA Ligase Dependente de ATP/metabolismo , DNA Ligase Dependente de ATP/genética , Reparo do DNA , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Endonucleases Flap/metabolismo , Endonucleases Flap/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genéticaRESUMO
The BRCA1 carboxyl-terminal (BRCT) domain, an evolutionarily conserved structural motif, is ubiquitous in a multitude of proteins spanning prokaryotic and eukaryotic organisms. In Mycobacterium tuberculosis (Mtb), BRCT domain plays a pivotal role in the catalytic activity of the NAD+-dependent DNA ligase (LigA). LigA is pivotal in DNA replication, catalyzing the formation of phosphodiester bonds in Okazaki fragments and repairing single-strand breaks in damaged DNA, essential for the survival of Mtb. Structural and functional aspects of LigA unveil its character as a highly modular protein, undergoing substantial conformational changes during its catalytic cycle. Although the BRCT domain of Mtb LigA plays an essential role in DNA binding and protein-protein interactions, the precise mechanism of action remains poorly understood. Unravelling the structure of the BRCT domain holds the promise of advancing our understanding of this pivotal domain. Additionally, it will facilitate further exploration of the protein-protein interactions and enhance our understanding of inter domain interactions within LigA, specifically between BRCT and the Adenylation domain. In this study, we demonstrate the overexpression of the BRCT domain of Mtb LigA and conduct its analysis using solution NMR spectroscopy, revealing a well-folded structure and we present the nearly complete chemical shift assignments of both backbone and sidechains. In addition, a secondary structure prediction by TALOS N predicts BRCT consisting of 3 α-helices and 4 ß-sheets, closely resembling the typical structural topology of most BRCT domains.
Assuntos
Mycobacterium tuberculosis , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Estrutura Secundária de Proteína , DNA Ligase Dependente de ATP/química , DNA Ligase Dependente de ATP/metabolismo , DNA Ligases/química , DNA Ligases/metabolismoRESUMO
This study establishes the physiological role of Fused in Sarcoma (FUS) in mitochondrial DNA (mtDNA) repair and highlights its implications to the pathogenesis of FUS-associated neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Endogenous FUS interacts with and recruits mtDNA Ligase IIIα (mtLig3) to DNA damage sites within mitochondria, a relationship essential for maintaining mtDNA repair and integrity in healthy cells. Using ALS patient-derived FUS mutant cell lines, a transgenic mouse model, and human autopsy samples, we discovered that compromised FUS functionality hinders mtLig3's repair role, resulting in increased mtDNA damage and mutations. These alterations cause various manifestations of mitochondrial dysfunction, particularly under stress conditions relevant to disease pathology. Importantly, rectifying FUS mutations in patient-derived induced pluripotent cells (iPSCs) preserves mtDNA integrity. Similarly, targeted introduction of human DNA Ligase 1 restores repair mechanisms and mitochondrial activity in FUS mutant cells, suggesting a potential therapeutic approach. Our findings unveil FUS's critical role in mitochondrial health and mtDNA repair, offering valuable insights into the mechanisms underlying mitochondrial dysfunction in FUS-associated motor neuron disease.
Assuntos
Esclerose Lateral Amiotrófica , Doenças Mitocondriais , Doença dos Neurônios Motores , Proteína FUS de Ligação a RNA , Animais , Humanos , Camundongos , Esclerose Lateral Amiotrófica/metabolismo , DNA Mitocondrial/genética , Ligases/metabolismo , Camundongos Transgênicos , Doença dos Neurônios Motores/genética , Doença dos Neurônios Motores/metabolismo , Mutação , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/metabolismoRESUMO
Base excision repair (BER) involves the tightly coordinated function of DNA polymerase ß (polß) and DNA ligase I (LIG1) at the downstream steps. Our previous studies emphasize that defective substrate-product channeling, from gap filling by polß to nick sealing by LIG1, can lead to interruptions in repair pathway coordination. Yet, the molecular determinants that dictate accurate BER remains largely unknown. Here, we demonstrate that a lack of gap filling by polß leads to faulty repair events and the formation of deleterious DNA intermediates. We dissect how ribonucleotide challenge and cancer-associated mutations could adversely impact the ability of polß to efficiently fill the one nucleotide gap repair intermediate which subsequently results in gap ligation by LIG1, leading to the formation of single-nucleotide deletion products. Moreover, we demonstrate that LIG1 is not capable of discriminating against nick DNA containing a 3'-ribonucleotide, regardless of base-pairing potential or damage. Finally, AP-Endonuclease 1 (APE1) shows distinct substrate specificity for the exonuclease removal of 3'-mismatched bases and ribonucleotides from nick repair intermediate. Overall, our results reveal that unfilled gaps result in impaired coordination between polß and LIG1, defining a possible type of mutagenic event at the downstream steps where APE1 could provide a proofreading role to maintain BER efficiency.
Assuntos
DNA Ligase Dependente de ATP , DNA Polimerase beta , Reparo do DNA , DNA Polimerase beta/metabolismo , DNA Polimerase beta/genética , DNA Ligase Dependente de ATP/metabolismo , DNA Ligase Dependente de ATP/genética , Humanos , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA/metabolismo , DNA/genética , Dano ao DNA , DNA Ligases/metabolismo , DNA Ligases/genética , Reparo por ExcisãoRESUMO
Ligase IV is a key enzyme involved during DNA double-strand breaks (DSBs) repair through nonhomologous end joining (NHEJ). However, in contrast to Ligase IV deficient mouse cells, which are embryonic lethal, Ligase IV deficient human cells, including pre-B cells, are viable. Using CRISPR-Cas9 mediated genome editing, we have generated six different LIG4 mutants in cervical cancer and normal kidney epithelial cell lines. While the LIG4 mutant cells showed a significant reduction in NHEJ, joining mediated through microhomology-mediated end joining (MMEJ) and homologous recombination (HR) were significantly high. The reduced NHEJ joining activity was restored by adding purified Ligase IV/XRCC4. Accumulation of DSBs and reduced cell viability were observed in LIG4 mutant cells. LIG4 mutant cells exhibited enhanced sensitivity towards DSB-inducing agents such as ionizing radiation (IR) and etoposide. More importantly, the LIG4 mutant of cervical cancer cells showed increased sensitivity towards FDA approved drugs such as Carboplatin, Cisplatin, Paclitaxel, Doxorubicin, and Bleomycin used for cervical cancer treatment. These drugs, in combination with IR showed enhanced cancer cell death in the background of LIG4 gene mutation. Thus, our study reveals that mutation in LIG4 results in compromised NHEJ, leading to sensitization of cervical cancer cells towards currently used cancer therapeutics.
Assuntos
DNA Ligase Dependente de ATP , Neoplasias do Colo do Útero , Animais , Feminino , Humanos , Camundongos , Dano ao DNA/genética , Reparo do DNA por Junção de Extremidades , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/metabolismo , DNA Ligases/genética , DNA Ligases/metabolismo , Reparo do DNA/genética , Ligases/genética , Ligases/metabolismo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismoRESUMO
The genome sequence of hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_0039, which encodes a putative DNA ligase. Structural analysis disclosed the presence of signature sequences of ATP-dependent DNA ligases. We have heterologously expressed Pcal_0039 gene in Escherichia coli. The recombinant protein, majorly produced in soluble form, was purified and functionally characterized. Recombinant Pcal_0039 displayed nick-joining activity between 40 and 85 °C. Optimal activity was observed at 70 °C and pH 5.5. Nick-joining activity was retained even after heating for 1 h at 90 °C, indicating highly thermostable nature of Pcal_0039. The nick-joining activity, displayed by Pcal_0039, was metal ion dependent and Mg2+ was the most preferred. NaCl and KCl inhibited the nick-joining activity at or above 200 mmol/L. The activity catalyzed by recombinant Pcal_0039 was independent of addition of ATP or NAD+ or any other nucleotide cofactor. A mismatch adjacent to the nick, either at 3'- or 5'-end, abolished the nick-joining activity. These characteristics make Pcal_0039 a potential candidate for applications in DNA diagnostics. To the best of our knowledge, Pcal_0039 is the only DNA ligase, characterized from genus Pyrobaculum, which exhibits optimum nick-joining activity at pH below 6.0 and independent of any nucleotide cofactor.
Assuntos
Pyrobaculum , Pyrobaculum/genética , NAD/metabolismo , Estabilidade Enzimática , DNA Ligase Dependente de ATP/metabolismo , DNA Ligases/genética , DNA Ligases/metabolismo , Archaea/metabolismo , Clonagem Molecular , Trifosfato de Adenosina/metabolismoRESUMO
Double-strand DNA breaks (DSBs) are toxic to cells, and improper repair can cause chromosomal abnormalities that initiate and drive cancer progression. DNA ligases III and IV (LIG3, LIG4) have long been credited for repair of DSBs in mammals, but recent evidence suggests that DNA ligase I (LIG1) has intrinsic end-joining (EJ) activity that can compensate for their loss. To test this model, we employed in vitro biochemical assays to compare EJ by LIG1 and LIG3. The ligases join blunt-end and 3'-overhang-containing DNA substrates with similar catalytic efficiency, but LIG1 joins 5'-overhang-containing DNA substrates â¼20-fold less efficiently than LIG3 under optimal conditions. LIG1-catalyzed EJ is compromised at a physiological concentration of Mg2+, but its activity is restored by increased molecular crowding. In contrast to LIG1, LIG3 efficiently catalyzes EJ reactions at a physiological concentration of Mg2+ with or without molecular crowding. Under all tested conditions, LIG3 has greater affinity than LIG1 for DNA ends. Remarkably, LIG3 can ligate both strands of a DSB during a single binding encounter. The weaker DNA binding affinity of LIG1 causes significant abortive ligation that is sensitive to molecular crowding and DNA terminal structure. These results provide new insights into mechanisms of alternative nonhomologous EJ.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Ligase Dependente de ATP , Reparo do DNA , Animais , Humanos , Reparo do DNA por Junção de Extremidades , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/metabolismo , Magnésio , Mamíferos/metabolismoRESUMO
BACKGROUND: DNA ligases are crucial for DNA repair and cell replication since they catalyze the final steps in which DNA breaks are joined. DNA Ligase III (LIG3) exerts a pivotal role in Alternative-Non-Homologous End Joining Repair (Alt-NHEJ), an error-prone DNA repair pathway often up-regulated in genomically unstable cancer, such as Multiple Myeloma (MM). Based on the three-dimensional (3D) LIG3 structure, we performed a computational screening to identify LIG3-targeting natural compounds as potential candidates to counteract Alt-NHEJ activity in MM. METHODS: Virtual screening was conducted by interrogating the Phenol Explorer database. Validation of binding to LIG3 recombinant protein was performed by Saturation Transfer Difference (STD)-nuclear magnetic resonance (NMR) experiments. Cell viability was analyzed by Cell Titer-Glo assay; apoptosis was evaluated by flow cytometric analysis following Annexin V-7AAD staining. Alt-NHEJ repair modulation was evaluated using plasmid re-joining assay and Cytoscan HD. DNA Damage Response protein levels were analyzed by Western blot of whole and fractionated protein extracts and immunofluorescence analysis. The mitochondrial DNA (mtDNA) copy number was determined by qPCR. In vivo activity was evaluated in NOD-SCID mice subcutaneously engrafted with MM cells. RESULTS: Here, we provide evidence that a natural flavonoid Rhamnetin (RHM), selected by a computational approach, counteracts LIG3 activity and killed Alt-NHEJ-dependent MM cells. Indeed, Nuclear Magnetic Resonance (NMR) showed binding of RHM to LIG3 protein and functional experiments revealed that RHM interferes with LIG3-driven nuclear and mitochondrial DNA repair, leading to significant anti-MM activity in vitro and in vivo. CONCLUSION: Taken together, our findings provide proof of concept that RHM targets LIG3 addiction in MM and may represent therefore a novel promising anti-tumor natural agent to be investigated in an early clinical setting.
Assuntos
DNA Ligase Dependente de ATP , Reparo do DNA , Flavonoides , Mieloma Múltiplo , Animais , Camundongos , Anexina A5/genética , Anexina A5/metabolismo , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/metabolismo , DNA Ligases/química , DNA Ligases/genética , DNA Ligases/metabolismo , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , DNA Mitocondrial/efeitos dos fármacos , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Flavonoides/farmacologia , Flavonoides/uso terapêutico , Camundongos Endogâmicos NOD , Camundongos SCID , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Fenóis , Proteínas Recombinantes/metabolismoRESUMO
The accumulation of epigenetic alterations is one of the major causes of tumorigenesis. Aberrant DNA methylation patterns cause genome instability and silencing of tumor suppressor genes in various types of tumors. Therefore, drugs that target DNA methylation-regulating factors have great potential for cancer therapy. Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1) is an essential factor for DNA methylation maintenance. UHRF1 is overexpressed in various cancer cells and down-regulation of UHRF1 in these cells reactivates the expression of tumor suppressor genes, thus UHRF1 is a promising target for cancer therapy. We have previously shown that interaction between the tandem Tudor domain (TTD) of UHRF1 and DNA ligase 1 (LIG1) di/trimethylated on Lys126 plays a key role in the recruitment of UHRF1 to replication sites and replication-coupled DNA methylation maintenance. An arginine binding cavity (Arg-binding cavity) of the TTD is essential for LIG1 interaction, thus the development of inhibitors that target the Arg-binding cavity could potentially repress UHRF1 function in cancer cells. To develop such an inhibitor, we performed in silico screening using not only static but also dynamic metrics based on all-atom molecular dynamics simulations, resulting in efficient identification of 5-amino-2,4-dimethylpyridine (5A-DMP) as a novel TTD-binding compound. Crystal structure of the TTD in complex with 5A-DMP revealed that the compound stably bound to the Arg-binding cavity of the TTD. Furthermore, 5A-DMP inhibits the full-length UHRF1:LIG1 interaction in Xenopus egg extracts. Our study uncovers a UHRF1 inhibitor which can be the basis of future experiments for cancer therapy.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/antagonistas & inibidores , DNA Ligase Dependente de ATP/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Simulação de Dinâmica Molecular , Piridinas/farmacologia , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , DNA Ligase Dependente de ATP/metabolismo , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Piridinas/química , Relação Estrutura-Atividade , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , XenopusRESUMO
Mutual crosstalk among poly(ADP-ribose) (PAR), activated PAR polymerase 1 (PARP1) metabolites, and DNA repair machinery has emerged as a key regulatory mechanism of the DNA damage response (DDR). However, there is no conclusive evidence of how PAR precisely controls DDR. Herein, six deubiquitinating enzymes (DUBs) associated with PAR-coupled DDR were identified, and the role of USP39, an inactive DUB involved in spliceosome assembly, was characterized. USP39 rapidly localizes to DNA lesions in a PAR-dependent manner, where it regulates non-homologous end-joining (NHEJ) via a tripartite RG motif located in the N-terminus comprising 46 amino acids (N46). Furthermore, USP39 acts as a molecular trigger for liquid demixing in a PAR-coupled N46-dependent manner, thereby directly interacting with the XRCC4/LIG4 complex during NHEJ. In parallel, the USP39-associated spliceosome complex controls homologous recombination repair in a PAR-independent manner. These findings provide mechanistic insights into how PAR chains precisely control DNA repair processes in the DDR.
Assuntos
Reparo do DNA por Junção de Extremidades , DNA Ligase Dependente de ATP/genética , Proteínas de Ligação a DNA/genética , DNA/genética , Poli(ADP-Ribose) Polimerases/genética , Proteases Específicas de Ubiquitina/genética , Motivos de Aminoácidos , Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Ligase Dependente de ATP/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Osteoblastos/citologia , Osteoblastos/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Reparo de DNA por Recombinação , Transdução de Sinais , Spliceossomos , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Proteases Específicas de Ubiquitina/metabolismoRESUMO
PURPOSE: Nonhomologous end-joining (NHEJ) is critical for the repair of either pathologic double-strand breaks (DSBs) and/or for the repair of physiologic DSBs created during radiotherapy to kill the tumor cell. Therefore, patients with higher expression of NHEJ repair proteins might develop resistance to ionizing radiation, allowing the disease to recur. As cancer of the oral cavity is a serious health problem globally, the present study aimed to examine the expression of Ku70/80, X-ray repair cross-complementing protein 4 (XRCC4) and DNA ligase IV-core molecules of the NHEJ pathway in patients with oral cancer. MATERIALS AND METHODS: Protein expression of Ku70/80, XRCC4, and DNA ligase IV were studied by Immunohistochemistry and mRNA expression of Ku70 and Ku80 were studied using reverse transcription polymerase chain reaction. Data were analyzed statistically using SPSS. RESULTS: A univariate survival analysis revealed an association of Ku70 mRNA with shorter overall survival (OS). While protein expression of XRCC4 showed an association with reduced relapse-free survival and shorter OS. Multivariate survival analysis demonstrated that XRCC4 and DNA ligase IV are independent prognosticators for predicting adverse disease outcomes. CONCLUSION: Strong expression of repair proteins - XRCC4 and DNA ligase IV is associated with unfavorable disease outcome in patients with oral squamous cell carcinoma.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/patologia , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Neoplasias Bucais/patologia , Recidiva Local de Neoplasia/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/terapia , Terapia Combinada , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Seguimentos , Humanos , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Neoplasias Bucais/terapia , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/terapia , Prognóstico , Taxa de Sobrevida , Adulto JovemRESUMO
Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, preclinical and clinical research with PARPi has revealed multiple resistance mechanisms, highlighting the need for identification of novel functional biomarkers and combination treatment strategies. Functional genetic screens performed in cells and organoids that acquired resistance to PARPi by loss of 53BP1 identified loss of LIG3 as an enhancer of PARPi toxicity in BRCA1-deficient cells. Enhancement of PARPi toxicity by LIG3 depletion is dependent on BRCA1 deficiency but independent of the loss of 53BP1 pathway. Mechanistically, we show that LIG3 loss promotes formation of MRE11-mediated post-replicative ssDNA gaps in BRCA1-deficient and BRCA1/53BP1 double-deficient cells exposed to PARPi, leading to an accumulation of chromosomal abnormalities. LIG3 depletion also enhances efficacy of PARPi against BRCA1-deficient mammary tumors in mice, suggesting LIG3 as a potential therapeutic target.
Assuntos
Proteína BRCA1/genética , DNA Ligase Dependente de ATP/genética , DNA de Cadeia Simples , Proteína Homóloga a MRE11/genética , Neoplasias Ovarianas/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Animais , Biópsia , Sistemas CRISPR-Cas , Linhagem Celular , Núcleo Celular/metabolismo , Proliferação de Células , Aberrações Cromossômicas , Dano ao DNA , DNA Ligase Dependente de ATP/metabolismo , Feminino , Humanos , Lentivirus/genética , Neoplasias Mamárias Animais , Camundongos , Mutação , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Interferente Pequeno/metabolismo , TransgenesRESUMO
Rationale: The human ligases (LIG1, LIG3 and LIG4) are essential for the maintenance of genomic integrity by catalysing the formation of phosphodiester bonds between adjacent 5'-phosphoryl and 3'-hydroxyl termini at single and double strand breaks in duplex DNA molecules generated either directly by DNA damage or during replication, recombination, and DNA repair. Whether LIG1, LIG3 and LIG4 can influence ovarian cancer pathogenesis and therapeutics is largely unknown. Methods: We investigated LIG1, LIG3 and LIG4 expression in clinical cohorts of epithelial ovarian cancers [protein level (n=525) and transcriptional level (n=1075)] and correlated to clinicopathological features and survival outcomes. Pre-clinically, platinum sensitivity was investigated in LIG1 depleted ovarian cancer cells. A small molecule inhibitor of LIG1 (L82) was tested for synthetic lethality application in XRCC1, BRCA2 or ATM deficient cancer cells. Results: LIG1 and LIG3 overexpression linked with aggressive phenotypes, platinum resistance and poor progression free survival (PFS). In contrast, LIG4 deficiency was associated with platinum resistance and worse PFS. In a multivariate analysis, LIG1 was independently associated with adverse outcome. In ovarian cancer cell lines, LIG1 depletion increased platinum cytotoxicity. L82 monotherapy was synthetically lethal in XRCC1 deficient ovarian cancer cells and 3D-spheroids. Increased cytotoxicity was linked with accumulation of DNA double strand breaks (DSBs), S-phase cell cycle arrest and increased apoptotic cells. L82 was also selectively toxic in BRCA2 deficient or ATM deficient cancer cells and 3D-spheroids. Conclusions: We provide evidence that LIG1 is an attractive target for personalization of ovarian cancer therapy.
Assuntos
Carcinoma Epitelial do Ovário/genética , DNA Ligase Dependente de ATP/metabolismo , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo , Adulto , Carcinoma Epitelial do Ovário/patologia , Linhagem Celular Tumoral , Cisplatino/farmacologia , DNA Ligase Dependente de ATP/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Ligases/genética , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Prognóstico , Transcriptoma/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genéticaRESUMO
The base excision repair (BER) pathway involves gap filling by DNA polymerase (pol) ß and subsequent nick sealing by ligase IIIα. X-ray cross-complementing protein 1 (XRCC1), a nonenzymatic scaffold protein, assembles multiprotein complexes, although the mechanism by which XRCC1 orchestrates the final steps of coordinated BER remains incompletely defined. Here, using a combination of biochemical and biophysical approaches, we revealed that the polß/XRCC1 complex increases the processivity of BER reactions after correct nucleotide insertion into gaps in DNA and enhances the handoff of nicked repair products to the final ligation step. Moreover, the mutagenic ligation of nicked repair intermediate following polß 8-oxodGTP insertion is enhanced in the presence of XRCC1. Our results demonstrated a stabilizing effect of XRCC1 on the formation of polß/dNTP/gap DNA and ligase IIIα/ATP/nick DNA catalytic ternary complexes. Real-time monitoring of protein-protein interactions and DNA-binding kinetics showed stronger binding of XRCC1 to polß than to ligase IIIα or aprataxin, and higher affinity for nick DNA with undamaged or damaged ends than for one nucleotide gap repair intermediate. Finally, we demonstrated slight differences in stable polß/XRCC1 complex formation, polß and ligase IIIα protein interaction kinetics, and handoff process as a result of cancer-associated (P161L, R194W, R280H, R399Q, Y576S) and cerebellar ataxia-related (K431N) XRCC1 variants. Overall, our findings provide novel insights into the coordinating role of XRCC1 and the effect of its disease-associated variants on substrate-product channeling in multiprotein/DNA complexes for efficient BER.
Assuntos
DNA Ligase Dependente de ATP/metabolismo , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo , DNA Polimerase beta/metabolismo , Reparo do DNA , Humanos , Cinética , Ligação Proteica , Ressonância de Plasmônio de SuperfícieRESUMO
Rac GTPase activating protein 1 (RACGAP1) has been characterized in the pathogenesis and progression of several malignancies, however, little is known regarding its role in the development of gallbladder cancer (GBC). This investigation seeks to describe the role of RACGAP1 and its associated molecular mechanisms in GBC. It was found that RACGAP1 was highly expressed in human GBC tissues, which was associated to poorer overall survival (OS). Gene knockdown of RACGAP1 hindered tumor cell proliferation and survival both in vitro and in vivo. We further identified that RACGAP1 was involved in DNA repair through its binding with DNA ligase 3 (LIG3), a crucial component of the alternative-non-homologous end joining (Alt-NHEJ) pathway. RACGAP1 regulated LIG3 expression independent of RhoA activity. RACGAP1 knockdown resulted in LIG3-dependent repair dysfunction, accumulated DNA damage and Poly(ADP-ribosyl) modification (PARylation) enhancement, leading to increased apoptosis and suppressed cell growth. We conclude that RACGAP1 exerts a tumor-promoting role via binding LIG3 to reduce apoptosis and facilitate cell growth in GBC, pointing to RACGAP1 as a potential therapeutic target for GBC.
Assuntos
Apoptose/genética , DNA Ligase Dependente de ATP/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Neoplasias da Vesícula Biliar/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Animais , Proliferação de Células/genética , DNA Ligase Dependente de ATP/genética , Feminino , Proteínas Ativadoras de GTPase/genética , Neoplasias da Vesícula Biliar/genética , Neoplasias da Vesícula Biliar/patologia , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Proteínas de Ligação a Poli-ADP-Ribose/genética , Taxa de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Nonhomologous end joining (NHEJ), one of the major DNA double-strand break repair pathways, plays a significant role in cancer cell proliferation and resistance to radio and chemotherapeutic agents. Previously, we had described a small molecule inhibitor, SCR7, which inhibited NHEJ in a DNA Ligase IV dependent manner. Here, we report that SCR7 potentiates the effect of γ-radiation (IR) that induces DNA breaks as intermediates to eradicate cancer cells. Dose fractionation studies revealed that coadministration of SCR7 and IR (0.5 Gy) in mice Dalton's lymphoma (DLA) model led to a significant reduction in mice tumor cell proliferation, which was equivalent to that observed for 2 Gy dose when both solid and liquid tumor models were used. Besides, co-treatment with SCR7 and 1 Gy of IR further improved the efficacy. Notably, there was no significant change in blood parameters, kidney and liver functions upon combinatorial treatment of SCR7 and IR. Further, the co-treatment of SCR7 and IR resulted in a significant increase in unrepaired DSBs within cancer cells compared to either of the agent alone. Anatomy, histology, and other studies in tumor models confirmed the cumulative effects of both agents in activating apoptotic pathways to induce cytotoxicity by modulating DNA damage response and repair pathways. Thus, we report that SCR7 has the potential to reduce the side effects of radiotherapy by lowering its effective dose ex vivo and in mice tumor models, with implications in cancer therapy.
Assuntos
Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/efeitos da radiação , Pirimidinas/farmacologia , Radiação Ionizante , Radiossensibilizantes/farmacologia , Bases de Schiff/farmacologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , DNA Ligase Dependente de ATP/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Chronic myeloid leukemia (CML) is characterized by the formation of the BCR-ABL fusion gene. The BCR-ABL protein leads to an increased level of reactive oxygen species, which is a major cause of endogenous DNA double-strand breaks (DSBs). CML cells are prone to rely on a highly mutagenic alternative end-joining (Alt-EJ) pathway to cope with enhanced DSBs, which aggravates chromosomal instability. Hence, targeting dysregulated DNA repair proteins provides new insights into cancer treatment. In this study, we discovered the abnormal upregulation of Flap endonuclease 1 (FEN1) in CML, as well as FEN1's participation in the error-prone Alt-EJ repair pathway and its interplay with DNA Ligase1 and proliferating cell nuclear antigen in DSB repair. Knockdown of FEN1 by shRNA not only inhibited the proliferation and induced apoptosis but also enhanced the efficacy of imatinib (IM) in drug-resistant CML cell K562/G01. Moreover, excessive DSB accumulation was detected after FEN1 inhibition. In summary, our results demonstrated that FEN1 is a promising therapeutic target in CML treatment. This work extends the understanding of regulating abnormal DSB repair for cancer treatment.
Assuntos
Proliferação de Células , Reparo do DNA por Junção de Extremidades , Endonucleases Flap/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Antineoplásicos/toxicidade , Apoptose , Células Cultivadas , DNA Ligase Dependente de ATP/metabolismo , Resistencia a Medicamentos Antineoplásicos , Endonucleases Flap/metabolismo , Células HEK293 , Humanos , Mesilato de Imatinib/toxicidade , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Células THP-1RESUMO
Evolutionary selection ensures specificity and efficiency in dynamic metastable macromolecular machines that repair DNA damage without releasing toxic and mutagenic intermediates. Here we examine non-homologous end joining (NHEJ) as the primary conserved DNA double-strand break (DSB) repair process in human cells. NHEJ has exemplary key roles in networks determining the development, outcome of cancer treatments by DSB-inducing agents, generation of antibody and T-cell receptor diversity, and innate immune response for RNA viruses. We determine mechanistic insights into NHEJ structural biochemistry focusing upon advanced small angle X-ray scattering (SAXS) results combined with X-ray crystallography (MX) and cryo-electron microscopy (cryo-EM). SAXS coupled to atomic structures enables integrated structural biology for objective quantitative assessment of conformational ensembles and assemblies in solution, intra-molecular distances, structural similarity, functional disorder, conformational switching, and flexibility. Importantly, NHEJ complexes in solution undergo larger allosteric transitions than seen in their cryo-EM or MX structures. In the long-range synaptic complex, X-ray repair cross-complementing 4 (XRCC4) plus XRCC4-like-factor (XLF) form a flexible bridge and linchpin for DNA ends bound to KU heterodimer (Ku70/80) and DNA-PKcs (DNA-dependent protein kinase catalytic subunit). Upon binding two DNA ends, auto-phosphorylation opens DNA-PKcs dimer licensing NHEJ via concerted conformational transformations of XLF-XRCC4, XLF-Ku80, and LigIVBRCT -Ku70 interfaces. Integrated structures reveal multifunctional roles for disordered linkers and modular dynamic interfaces promoting DSB end processing and alignment into the short-range complex for ligation by LigIV. Integrated findings define dynamic assemblies fundamental to designing separation-of-function mutants and allosteric inhibitors targeting conformational transitions in multifunctional complexes.