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1.
Dev Cell ; 56(4): 461-477.e7, 2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33621493

RESUMO

Homology-directed repair (HDR) safeguards DNA integrity under various forms of stress, but how HDR protects replicating genomes under extensive metabolic alterations remains unclear. Here, we report that besides stalling replication forks, inhibition of ribonucleotide reductase (RNR) triggers metabolic imbalance manifested by the accumulation of increased reactive oxygen species (ROS) in cell nuclei. This leads to a redox-sensitive activation of the ATM kinase followed by phosphorylation of the MRE11 nuclease, which in HDR-deficient settings degrades stalled replication forks. Intriguingly, nascent DNA degradation by the ROS-ATM-MRE11 cascade is also triggered by hypoxia, which elevates signaling-competent ROS and attenuates functional HDR without arresting replication forks. Under these conditions, MRE11 degrades daughter-strand DNA gaps, which accumulate behind active replisomes and attract error-prone DNA polymerases to escalate mutation rates. Thus, HDR safeguards replicating genomes against metabolic assaults by restraining mutagenic repair at aberrantly processed nascent DNA. These findings have implications for cancer evolution and tumor therapy.


Assuntos
Replicação do DNA , Genoma Humano , Metabolismo , Reparo de DNA por Recombinação , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA2/deficiência , Proteína BRCA2/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , DNA/metabolismo , Humanos , Proteína Homóloga a MRE11/metabolismo , Modelos Biológicos , Mutação/genética , Neoplasias/genética , Neoplasias/patologia , Polimerização , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
2.
Nature ; 587(7833): 297-302, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33087936

RESUMO

Minichromosome maintenance proteins (MCMs) are DNA-dependent ATPases that bind to replication origins and license them to support a single round of DNA replication. A large excess of MCM2-7 assembles on chromatin in G1 phase as pre-replication complexes (pre-RCs), of which only a fraction become the productive CDC45-MCM-GINS (CMG) helicases that are required for genome duplication1-4. It remains unclear why cells generate this surplus of MCMs, how they manage to sustain it across multiple generations, and why even a mild reduction in the MCM pool compromises the integrity of replicating genomes5,6. Here we show that, for daughter cells to sustain error-free DNA replication, their mother cells build up a nuclear pool of MCMs both by recycling chromatin-bound (parental) MCMs and by synthesizing new (nascent) MCMs. Although all MCMs can form pre-RCs, it is the parental pool that is inherently stable and preferentially matures into CMGs. By contrast, nascent MCM3-7 (but not MCM2) undergo rapid proteolysis in the cytoplasm, and their stabilization and nuclear translocation require interaction with minichromosome-maintenance complex-binding protein (MCMBP), a distant MCM paralogue7,8. By chaperoning nascent MCMs, MCMBP safeguards replicating genomes by increasing chromatin coverage with pre-RCs that do not participate on replication origins but adjust the pace of replisome movement to minimize errors during DNA replication. Consequently, although the paucity of pre-RCs in MCMBP-deficient cells does not alter DNA synthesis overall, it increases the speed and asymmetry of individual replisomes, which leads to DNA damage. The surplus of MCMs therefore increases the robustness of genome duplication by restraining the speed at which eukaryotic cells replicate their DNA. Alterations in physiological fork speed might thus explain why even a minor reduction in MCM levels destabilizes the genome and predisposes to increased incidence of tumour formation.


Assuntos
Replicação do DNA/genética , Genoma Humano/genética , Proteínas de Manutenção de Minicromossomo/biossíntese , Proteínas de Manutenção de Minicromossomo/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Dano ao DNA , Humanos , Proteínas de Manutenção de Minicromossomo/análise , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Estabilidade Proteica , Transporte Proteico
4.
Cell Rep ; 30(7): 2416-2429.e7, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32075739

RESUMO

It has been long assumed that normally leading strand synthesis must proceed coordinated with the lagging strand to prevent strand uncoupling and the pathological accumulation of single-stranded DNA (ssDNA) in the cell, a dogma recently challenged by in vitro studies in prokaryotes. Here, we report that human DNA polymerases can function independently at each strand in vivo and that the resulting strand uncoupling is supported physiologically by a cellular tolerance to ssDNA. Active forks rapidly accumulate ssDNA at the lagging strand when POLA1 is inhibited without triggering a stress response, despite ssDNA formation being considered a hallmark of replication stress. Acute POLA1 inhibition causes a lethal RPA exhaustion, but cells can duplicate their DNA with limited POLA1 activity and exacerbated strand uncoupling as long as RPA molecules suffice to protect the elevated ssDNA. Although robust, this uncoupled mode of DNA replication is also an in-built weakness that can be targeted for cancer treatment.


Assuntos
Replicação do DNA/genética , DNA de Cadeia Simples/genética , Ligação Proteica/genética , Humanos
5.
Cell Rep ; 29(3): 551-559.e4, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31618626

RESUMO

ATR kinase-mediated replication checkpoint is vital for genome maintenance following replication stress. Previously, we showed that XRCC2-RAD51D (DX2) sub-complex of RAD51 paralogs restrains active DNA synthesis during dNTP alterations, in a manner dependent on ATR-mediated phosphorylation of XRCC2. Here, we find that unrestrained fork progression in XRCC2 deficiency and phosphorylation defect causes replication-associated errors, subsequently resulting in genome-wide double-strand breaks (DSBs) and early activation of ATM signaling. Cells defective in XRCC2 phosphorylation exhibit ATM/ATR-mediated early activation of XRCC3 during perturbed replication, which facilitates recombination-mediated repair of the post-replicative DNA damage and thereby promotes cell viability. Collectively, our findings identify collaborative roles of RAD51 paralog complexes during replication stress and reveal their differential regulation by ATR signaling to promote cell survival and genome integrity.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Replicação do DNA , Recombinação Homóloga , Rad51 Recombinase/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica , Humanos , Hidroxiureia/farmacologia , Morfolinas/farmacologia , Mutagênese Sítio-Dirigida , Fosforilação , Pironas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Rad51 Recombinase/genética , Transdução de Sinais
7.
Nat Cell Biol ; 21(4): 487-497, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30804506

RESUMO

Failure to complete DNA replication is a stochastic by-product of genome doubling in almost every cell cycle. During mitosis, under-replicated DNA (UR-DNA) is converted into DNA lesions, which are inherited by daughter cells and sequestered in 53BP1 nuclear bodies (53BP1-NBs). The fate of such cells remains unknown. Here, we show that the formation of 53BP1-NBs interrupts the chain of iterative damage intrinsically embedded in UR-DNA. Unlike clastogen-induced 53BP1 foci that are repaired throughout interphase, 53BP1-NBs restrain replication of the embedded genomic loci until late S phase, thus enabling the dedicated RAD52-mediated repair of UR-DNA lesions. The absence or malfunction of 53BP1-NBs causes premature replication of the affected loci, accompanied by genotoxic RAD51-mediated recombination. Thus, through adjusting replication timing and repair pathway choice at under-replicated loci, 53BP1-NBs enable the completion of genome duplication of inherited UR-DNA and prevent the conversion of stochastic under-replications into genome instability.


Assuntos
Estruturas do Núcleo Celular/fisiologia , Dano ao DNA , Período de Replicação do DNA , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/fisiologia , Linhagem Celular , Segregação de Cromossomos , Reparo do DNA , Replicação do DNA , Humanos , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinação Genética , Fase S/genética , Proteínas de Ligação a Telômeros/fisiologia
8.
Cell Rep ; 25(12): 3273-3282.e6, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30566856

RESUMO

RAD51 paralogs are essential for maintenance of genomic integrity through protection of stalled replication forks and homology-directed repair (HDR) of double-strand breaks. Here, we find that a subset of RAD51 paralogs, XRCC2 (FANCU) and its binding partner RAD51D, restrain active DNA synthesis during dinucleotide triphosphate (dNTP) alterations in a manner independent of HDR. The absence of XRCC2 is associated with increased levels of RRM2, the regulatory subunit of ribonucleotide reductase (RNR), and concomitantly high nucleotide pools, leading to unrestrained fork progression and accumulation of DNA damage during dNTP alterations. Mechanistically, this function is independent of redox signaling and RAD51-mediated fork reversal and is regulated by ataxia-telangiectasia and Rad3-related (ATR) signaling through phosphorylation of XRCC2 (Ser247). Together, these findings identify roles of RAD51 paralogs in the control of replication fork progression and maintenance of genome stability during nucleotide pool alterations.


Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Nucleotídeos/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , DNA/biossíntese , Humanos , Cinética , Modelos Biológicos , Oxirredução , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Ribonucleotídeo Redutases/metabolismo , Transdução de Sinais , Estresse Fisiológico
9.
Cell ; 173(4): 972-988.e23, 2018 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-29656893

RESUMO

Repair of damaged DNA is essential for maintaining genome integrity and for preventing genome-instability-associated diseases, such as cancer. By combining proximity labeling with quantitative mass spectrometry, we generated high-resolution interaction neighborhood maps of the endogenously expressed DNA repair factors 53BP1, BRCA1, and MDC1. Our spatially resolved interaction maps reveal rich network intricacies, identify shared and bait-specific interaction modules, and implicate previously concealed regulators in this process. We identified a novel vertebrate-specific protein complex, shieldin, comprising REV7 plus three previously uncharacterized proteins, RINN1 (CTC-534A2.2), RINN2 (FAM35A), and RINN3 (C20ORF196). Recruitment of shieldin to DSBs, via the ATM-RNF8-RNF168-53BP1-RIF1 axis, promotes NHEJ-dependent repair of intrachromosomal breaks, immunoglobulin class-switch recombination (CSR), and fusion of unprotected telomeres. Shieldin functions as a downstream effector of 53BP1-RIF1 in restraining DNA end resection and in sensitizing BRCA1-deficient cells to PARP inhibitors. These findings have implications for understanding cancer-associated PARPi resistance and the evolution of antibody CSR in higher vertebrates.


Assuntos
Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas Adaptadoras de Transdução de Sinal , Proteína BRCA1/antagonistas & inibidores , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Humanos , Switching de Imunoglobulina/efeitos dos fármacos , Proteínas Mad2/antagonistas & inibidores , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Mutagênese Sítio-Dirigida , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a Telômeros/antagonistas & inibidores , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Transativadores/genética , Transativadores/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
10.
Science ; 358(6364): 797-802, 2017 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-29123070

RESUMO

DNA replication requires coordination between replication fork progression and deoxynucleotide triphosphate (dNTP)-generating metabolic pathways. We find that perturbation of ribonucleotide reductase (RNR) in humans elevates reactive oxygen species (ROS) that are detected by peroxiredoxin 2 (PRDX2). In the oligomeric state, PRDX2 forms a replisome-associated ROS sensor, which binds the fork accelerator TIMELESS when exposed to low levels of ROS. Elevated ROS levels generated by RNR attenuation disrupt oligomerized PRDX2 to smaller subunits, whose dissociation from chromatin enforces the displacement of TIMELESS from the replisome. This process instantly slows replication fork progression, which mitigates pathological consequences of replication stress. Thus, redox signaling couples fluctuations of dNTP biogenesis with replisome activity to reduce stress during genome duplication. We propose that cancer cells exploit this pathway to increase their adaptability to adverse metabolic conditions.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Instabilidade Genômica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias/genética , Peroxirredoxinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ribonucleotídeo Redutases/metabolismo , Adaptação Biológica , Cromatina/metabolismo , Desoxirribonucleotídeos/metabolismo , Humanos , Redes e Vias Metabólicas , Oxirredução , Transdução de Sinais
11.
Nucleic Acids Res ; 45(15): 8886-8900, 2017 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-28911102

RESUMO

The FANCJ DNA helicase is linked to hereditary breast and ovarian cancers as well as bone marrow failure disorder Fanconi anemia (FA). Although FANCJ has been implicated in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR), the molecular mechanism underlying the tumor suppressor functions of FANCJ remains obscure. Here, we demonstrate that FANCJ deficient human and hamster cells exhibit reduction in the overall gene conversions in response to a site-specific chromosomal DSB induced by I-SceI endonuclease. Strikingly, the gene conversion events were biased in favour of long-tract gene conversions in FANCJ depleted cells. The fine regulation of short- (STGC) and long-tract gene conversions (LTGC) by FANCJ was dependent on its interaction with BRCA1 tumor suppressor. Notably, helicase activity of FANCJ was essential for controlling the overall HR and in terminating the extended repair synthesis during sister chromatid recombination (SCR). Moreover, cells expressing FANCJ pathological mutants exhibited defective SCR with an increased frequency of LTGC. These data unravel the novel function of FANCJ helicase in regulating SCR and SCR associated gene amplification/duplications and imply that these functions of FANCJ are crucial for the genome maintenance and tumor suppression.


Assuntos
Proteína BRCA1/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Cromátides/química , DNA/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Reparo de DNA por Recombinação , Animais , Proteína BRCA1/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Células CHO , Linhagem Celular Tumoral , Cromátides/metabolismo , Cricetulus , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Desoxirribonucleases de Sítio Específico do Tipo II/farmacologia , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular , Regulação da Expressão Gênica , Recombinação Homóloga/efeitos dos fármacos , Humanos , Mutação , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/farmacologia
12.
Nat Struct Mol Biol ; 23(8): 714-21, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27348077

RESUMO

Repair of DNA double-strand breaks (DSBs) in mammals is coordinated by the ubiquitin-dependent accumulation of 53BP1 at DSB-flanking chromatin. Owing to its ability to limit DNA-end processing, 53BP1 is thought to promote nonhomologous end-joining (NHEJ) and to suppress homology-directed repair (HDR). Here, we show that silencing 53BP1 or exhausting its capacity to bind damaged chromatin changes limited DSB resection to hyper-resection and results in a switch from error-free gene conversion by RAD51 to mutagenic single-strand annealing by RAD52. Thus, rather than suppressing HDR, 53BP1 fosters its fidelity. These findings illuminate causes and consequences of synthetic viability acquired through 53BP1 silencing in cells lacking the BRCA1 tumor suppressor. We show that such cells survive DSB assaults at the cost of increasing reliance on RAD52-mediated HDR, which may fuel genome instability. However, our findings suggest that when challenged by DSBs, BRCA1- and 53BP1-deficient cells may become hypersensitive to, and be eliminated by, RAD52 inhibition.


Assuntos
Proteína 1 de Ligação à Proteína Supressora de Tumor p53/fisiologia , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Humanos , Transporte Proteico , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo
13.
Carcinogenesis ; 37(2): 145-156, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26678223

RESUMO

Although DNA interstrand crosslinking (ICL) agents such as mitomycin C, cisplatin and psoralen serve as potent anticancer drugs, these agents are known to have dose-limiting toxic effects on normal cells. Moreover, tumor resistance to these agents has been reported. Here, we show that trans-dichlorooxovanadium (IV) complex of pyrenyl terpyridine (VDC) is a novel photoinducible DNA crosslinking agent. By a combination of in vitro and ex vivo experiments including plasmid-based assays, we find that VDC forms monoadducts on the DNA and can be activated by UV-A and visible light to generate DNA interstrand crosslinks. VDC efficiently activates Fanconi anemia (FA) pathway of DNA interstrand crosslink repair. Strikingly, photoinduction of VDC induces prolonged activation of cell cycle checkpoint and a high degree of cell death in homologous recombination (HR)/ICL repair defective cells. Moreover, VDC specifically targets cells that express pathological RAD51C mutants. These data imply that VDC can be potentially used for cancer therapy and suggest that tumors arising in patients with gene mutations in FA and HR repair pathway can be specifically targeted by a photoactivatable VDC.


Assuntos
Antineoplásicos/farmacologia , Complexos de Coordenação/farmacocinética , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Neoplasias/patologia , Fármacos Fotossensibilizantes/farmacologia , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ensaio Cometa , Reagentes para Ligações Cruzadas/farmacologia , Adutos de DNA/efeitos dos fármacos , Imunofluorescência , Humanos , Neoplasias/genética
14.
Nucleic Acids Res ; 43(20): 9835-55, 2015 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-26354865

RESUMO

Mammalian RAD51 paralogs are implicated in the repair of collapsed replication forks by homologous recombination. However, their physiological roles in replication fork maintenance prior to fork collapse remain obscure. Here, we report on the role of RAD51 paralogs in short-term replicative stress devoid of DSBs. We show that RAD51 paralogs localize to nascent DNA and common fragile sites upon replication fork stalling. Strikingly, RAD51 paralogs deficient cells exhibit elevated levels of 53BP1 nuclear bodies and increased DSB formation, the latter being attributed to extensive degradation of nascent DNA at stalled forks. RAD51C and XRCC3 promote the restart of stalled replication in an ATP hydrolysis dependent manner by disengaging RAD51 and other RAD51 paralogs from the halted forks. Notably, we find that Fanconi anemia (FA)-like disorder and breast and ovarian cancer patient derived mutations of RAD51C fails to protect replication fork, exhibit under-replicated genomic regions and elevated micro-nucleation. Taken together, RAD51 paralogs prevent degradation of stalled forks and promote the restart of halted replication to avoid replication fork collapse, thereby maintaining genomic integrity and suppressing tumorigenesis.


Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/fisiologia , DNA/metabolismo , Motivos de Aminoácidos , Animais , Neoplasias da Mama/genética , Linhagem Celular , Cromatina/metabolismo , Sítios Frágeis do Cromossomo , Cricetinae , Cricetulus , Quebras de DNA , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Feminino , Células HeLa , Humanos , Complexos Multienzimáticos , Mutação , Neoplasias Ovarianas/genética , Fase S
15.
PLoS One ; 10(6): e0127558, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26083398

RESUMO

Thrombocytopenia in methotrexate (MTX)-treated cancer and rheumatoid arthritis (RA) patients connotes the interference of MTX with platelets. Hence, it seemed appealing to appraise the effect of MTX on platelets. Thereby, the mechanism of action of MTX on platelets was dissected. MTX (10 µM) induced activation of pro-apoptotic proteins Bid, Bax and Bad through JNK phosphorylation leading to ΔΨm dissipation, cytochrome c release and caspase activation, culminating in apoptosis. The use of specific inhibitor for JNK abrogates the MTX-induced activation of pro-apoptotic proteins and downstream events confirming JNK phosphorylation by MTX as a key event. We also demonstrate that platelet mitochondria as prime sources of ROS which plays a central role in MTX-induced apoptosis. Further, MTX induces oxidative stress by altering the levels of ROS and glutathione cycle. In parallel, the clinically approved thiol antioxidant N-acetylcysteine (NAC) and its derivative N-acetylcysteine amide (NACA) proficiently alleviate MTX-induced platelet apoptosis and oxidative damage. These findings underpin the dearth of research on interference of therapeutic drugs with platelets, despite their importance in human health and disease. Therefore, the use of antioxidants as supplementary therapy seems to be a safe bet in pathologies associated with altered platelet functions.


Assuntos
Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Antimetabólitos Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , MAP Quinase Quinase 4/genética , Metotrexato/farmacologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Regulação da Expressão Gênica , Humanos , MAP Quinase Quinase 4/metabolismo , Potencial da Membrana Mitocondrial , Metotrexato/antagonistas & inibidores , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oxirredução , Estresse Oxidativo , Fosforilação , Cultura Primária de Células , Transdução de Sinais , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína de Morte Celular Associada a bcl/genética , Proteína de Morte Celular Associada a bcl/metabolismo
16.
Carcinogenesis ; 36(1): 13-24, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25292178

RESUMO

Poly (ADP-ribose) polymerase 1 (PARP1) inhibitors are actively under clinical trials for the treatment of breast and ovarian cancers that arise due to mutations in BRCA1 and BRCA2. The RAD51 paralog RAD51C has been identified as a breast and ovarian cancer susceptibility gene. The pathological RAD51C mutants that were identified in cancer patients are hypomorphic with partial repair function. However, targeting cancer cells that express hypomorphic mutants of RAD51C is highly challenging. Here, we report that RAD51C-deficient cells can be targeted by a 'synthetic lethal' approach using PARP inhibitor and this sensitivity was attributed to accumulation of cells in the G2/M and chromosomal aberrations. In addition, spontaneous hyperactivation of PARP1 was evident in RAD51C-deficient cells. Interestingly, RAD51C-negative cells exhibited enhanced recruitment of non-homologous end joining (NHEJ) proteins onto chromatin and this accumulation correlated with increased activity of error-prone NHEJ as well as genome instability leading to cell death. Notably, inhibition of DNA-PKcs or depletion of KU70 or Ligase IV rescued this phenotype. Strikingly, stimulation of NHEJ by low dose of ionizing radiation (IR) in the PARP inhibitor-treated RAD51C-deficient cells and cells expressing pathological RAD51C mutants induced enhanced toxicity 'synergistically'. These results demonstrate that cancer cells arising due to hypomorphic mutations in RAD51C can be specifically targeted by a 'synergistic approach' and imply that this strategy can be potentially applied to cancers with hypomorphic mutations in other homologous recombination pathway genes.


Assuntos
Neoplasias da Mama/patologia , Reparo do DNA por Junção de Extremidades/genética , Proteínas de Ligação a DNA/genética , Mutação/genética , Poli(ADP-Ribose) Polimerases/genética , Recombinação Genética , Western Blotting , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Ciclo Celular , Proliferação de Células , Cromatina/genética , Aberrações Cromossômicas , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Imunofluorescência , Instabilidade Genômica , Células HeLa , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/metabolismo , Células Tumorais Cultivadas
17.
Dalton Trans ; 43(35): 13358-69, 2014 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-25069796

RESUMO

Oxovanadium(IV) complexes [VO(R-tpy)(cur)](ClO4) (1, 2) of curcumin (Hcur) and terpyridine ligands (R-tpy) where R is phenyl (phtpy in 1) or p-triphenylphosphonium methylphenyl bromide (C6H4CH2PPh3Br) (TPP-phtpy in 2) were prepared and characterized and their DNA photocleavage activity, photocytotoxicity and cellular localization in cancer cells (HeLa and MCF-7) were studied. Acetylacetonate (acac) complexes [VO(R-tpy)(acac)](ClO4) of phtpy (3) and TPP-phtpy (4) were prepared and used as the control species. These complexes showed efficient cleavage of pUC19 DNA in visible light of 454 nm and near-IR light of 705 nm. Complexes 1 and 2 showed significant photocytotoxicity in visible light of 400-700 nm. FACS analysis showed sub-G1/G0 phase cell-cycle arrest in cancer cells when treated with 1 and 2 in visible light in comparison with the dark controls. Fluorescence microscopic studies revealed specific localization of the p-triphenylphosphonium complex 2 in the mitochondria of MCF-7 cancer cells whereas no such specificity was observed for complex 1.


Assuntos
Curcumina/química , Citotoxinas/química , Sistemas de Liberação de Medicamentos/métodos , Mitocôndrias/química , Vanadatos/química , Animais , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Curcumina/administração & dosagem , Citotoxinas/administração & dosagem , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Células MCF-7 , Microscopia Confocal/métodos , Mitocôndrias/efeitos dos fármacos , Estimulação Luminosa/métodos , Vanadatos/administração & dosagem
18.
Dalton Trans ; 43(3): 1321-31, 2014 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-24193217

RESUMO

Oxovanadium(IV) complexes [VO(aip)(L)](ClO4)2 (L = phtpy, 1; stpy, 2) and [VO(pyip)(L)](ClO4)2 (L = phtpy, 3; stpy, 4), where aip is 2-(9-anthryl)-1H-imidazo[4,5-f][1,10]phenanthroline, pyip is [2-(1-pyrenyl)-1H-imidazo[4,5-f][1,10]phenanthroline, phtpy is (4'-phenyl)-2,2':6',2''-terpyridine and stpy is (2,2':6',2''-terpyridin-4'-oxy)ethyl-ß-D-glucopyranoside, were prepared, characterized and their DNA binding and photocleavage activity, cellular uptake and photocytotoxicity in visible light were studied. The complexes are avid binders to calf thymus DNA (K(b) ~10(5) mol(-1)). They efficiently cleave pUC19 DNA in red light of 705 nm via the formation of HO˙ species. The glucose appended complexes 2 and 4 showed higher photocytotoxicity in HeLa and Hep G2 cells over the normal HEK 293T cells. No such preference was observed for the phtpy complexes 1 and 3. No significant difference in IC50 values was observed for the HEK 293T cells. Cell cycle analysis showed that the glucose appended complexes 2 and 4 are more photocytotoxic in cancer cells than in normal cells. Fluorescence microscopy was done to study the cellular localization of complex 4 having a pendant pyrenyl group.


Assuntos
Carboidratos/química , Complexos de Coordenação/química , Vanadatos/química , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/metabolismo , Complexos de Coordenação/farmacologia , DNA/química , DNA/metabolismo , Clivagem do DNA/efeitos dos fármacos , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Imidazóis/química , Luz , Microscopia Confocal , Fenantrolinas/química
19.
FEBS J ; 280(8): 1841-60, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23438087

RESUMO

In order to survive and replicate in a variety of stressful conditions during its life cycle, Mycobacterium tuberculosis must possess mechanisms to safeguard the integrity of the genome. Although DNA repair and recombination related genes are thought to play key roles in the repair of damaged DNA in all organisms, so far only a few of them have been functionally characterized in the tubercle bacillus. In this study, we show that M. tuberculosis RecG (MtRecG) expression was induced in response to different genotoxic agents. Strikingly, expression of MtRecG in Escherichia coli ∆recG mutant strain provided protection against mitomycin C, methyl methane sulfonate and UV induced cell death. Purified MtRecG exhibited higher binding affinity for the Holliday junction (HJ) compared with a number of canonical recombinational DNA repair intermediates. Notably, although MtRecG binds at the core of the mobile and immobile HJs, and with higher binding affinity for the immobile HJ, branch migration was evident only in the case of the mobile HJ. Furthermore, immobile HJs stimulate MtRecG ATPase activity less efficiently than mobile HJs. In addition to HJ substrates, MtRecG exhibited binding affinity for a variety of branched DNA structures including three-way junctions, replication forks, flap structures, forked duplex and a D-loop structure, but demonstrated strong unwinding activity on replication fork and flap DNA structures. Together, these results support that MtRecG plays an important role in processes related to DNA metabolism under normal as well as stress conditions.


Assuntos
DNA Helicases/fisiologia , Reparo do DNA , Mycobacterium tuberculosis/genética , Recombinação Genética , Trifosfato de Adenosina/metabolismo , Clonagem Molecular , Dano ao DNA
20.
Mol Cell Biol ; 33(9): 1830-44, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23438602

RESUMO

The RAD51 paralogs XRCC3 and RAD51C have been implicated in homologous recombination (HR) and DNA damage responses. However, the molecular mechanism(s) by which these paralogs regulate HR and DNA damage signaling remains obscure. Here, we show that an SQ motif serine 225 in XRCC3 is phosphorylated by ATR kinase in an ATM signaling pathway. We find that RAD51C but not XRCC2 is essential for XRCC3 phosphorylation, and this modification follows end resection and is specific to S and G2 phases. XRCC3 phosphorylation is required for chromatin loading of RAD51 and HR-mediated repair of double-strand breaks (DSBs). Notably, in response to DSBs, XRCC3 participates in the intra-S-phase checkpoint following its phosphorylation and in the G2/M checkpoint independently of its phosphorylation. Strikingly, we find that XRCC3 distinctly regulates recovery of stalled and collapsed replication forks such that phosphorylation is required for the HR-mediated recovery of collapsed replication forks but is dispensable for the restart of stalled replication forks. Together, these findings suggest that XRCC3 is a new player in the ATM/ATR-induced DNA damage responses to control checkpoint and HR-mediated repair.


Assuntos
Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia , Linhagem Celular , DNA/química , DNA/genética , Reparo do DNA , Proteínas de Ligação a DNA/química , Humanos , Interfase , Dados de Sequência Molecular , Fosforilação , Alinhamento de Sequência
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