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1.
Int J Mol Sci ; 25(7)2024 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-38612480

RESUMO

The aim of this study was to investigate gene expression alterations associated with overall survival (OS) in glioblastoma (GBM). Using the Nanostring nCounter platform, we identified four genes (COL1A2, IGFBP3, NGFR, and WIF1) that achieved statistical significance when comparing GBM with non-neoplastic brain tissue. The four genes were included in a multivariate Cox Proportional Hazard model, along with age, extent of resection, and O6-methylguanine-DNA methyltransferase (MGMT) promotor methylation, to create a unique glioblastoma prognostic index (GPI). The GPI score inversely correlated with survival: patient with a high GPI had a median OS of 7.5 months (18-month OS = 9.7%) whereas patients with a low GPI had a median OS of 20.1 months (18-month OS = 54.5%; log rank p-value = 0.004). The GPI score was then validated in 188 GBM patients from The Cancer Genome Atlas (TCGA) from a national data base; similarly, patients with a high GPI had a median OS of 10.5 months (18-month OS = 12.4%) versus 16.9 months (18-month OS = 41.5%) for low GPI (log rank p-value = 0.0003). We conclude that this novel mRNA-based prognostic index could be useful in classifying GBM patients into risk groups and refine prognosis estimates to better inform treatment decisions or stratification into clinical trials.


Assuntos
Glioblastoma , Humanos , Glioblastoma/genética , Genes Reguladores , Bases de Dados Factuais , O(6)-Metilguanina-DNA Metiltransferase , Expressão Gênica
2.
Cancers (Basel) ; 13(21)2021 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-34771522

RESUMO

Treatment failures of glioblastoma (GBM) occur within high-dose radiation fields. We hypothesized that this is due to increased capacity for DNA damage repair in GBM. We identified 24 adult GBM patients treated with maximal safe resection followed by radiation with concurrent and adjuvant temozolomide. The mRNA from patients was quantified using NanoString Technologies' nCounter platform and compared with 12 non-neoplastic temporal lobe tissue samples as a control. Differential expression analysis identified seven DNA repair genes significantly upregulated in GBM tissues relative to controls (>4-fold difference, adjusted p values < 0.001). Among these seven genes, Cox proportional hazards models identified RAD51 to be associated with an increased risk of death (HR = 3.49; p = 0.03). Kaplan-Meier (KM) analysis showed that patients with high RAD51 expression had significantly shorter OS compared to low levels (median OS of 10.6 mo. vs 20.1 mo.; log-rank p = 0.03). Our findings were validated in a larger external dataset of 162 patients using publicly available gene expression data quantified by the same NanoString technology (median OS of 13.8 mo. vs. 17.4 mo; log-rank p = 0.006). Within this uniformly treated GBM population, RAD51, in the homologous recombination pathway, was overexpressed (vs. normal brain) and inversely correlated with OS. High RAD51 expression may be a prognostic biomarker and a therapeutic target in GBM.

3.
Sci Rep ; 9(1): 18347, 2019 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-31797958

RESUMO

We investigated biomarker CEACAM6, a highly abundant cell surface adhesion receptor that modulates the extracellular matrix (ECM) in pancreatic ductal adenocarcinoma (PDA). The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) RNA-Seq data from PDA patients were analyzed for CEACAM6 expression and evaluated for overall survival, association, enrichment and correlations. A CRISPR/Cas9 Knockout (KO) of CEACAM6 in PDA cell line for quantitative proteomics, mitochondrial bioenergetics and tumor growth in mice were conducted. We found CEACAM6 is over-expressed in primary and metastatic basal and classical PDA subtypes. Highest levels are in classical activated stroma subtype. CEACAM6 over-expression is universally a poor prognostic marker in KRAS mutant and wild type PDA. High CEACAM6 expression is associated with low cytolytic T-cell activity in both basal and classical PDA subtypes and correlates with low levels of T-REG markers. In HPAF-II cells knockout of CEACAM6 alters ECM-cell adhesion, catabolism, immune environment, transmembrane transport and autophagy. CEACAM6 loss increases mitochondrial basal and maximal respiratory capacity. HPAF-II CEACAM6-/- cells are growth suppressed by >65% vs. wild type in mice bearing tumors. CEACAM6, a key regulator affects several hallmarks of PDA including the fibrotic reaction, immune regulation, energy metabolism and is a novel therapeutic target in PDA.


Assuntos
Adenocarcinoma/genética , Antígenos CD/genética , Carcinoma Ductal Pancreático/genética , Moléculas de Adesão Celular/genética , Linfócitos T/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma/terapia , Animais , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/terapia , Linhagem Celular Tumoral , Proliferação de Células/genética , Metabolismo Energético/genética , Proteínas Ligadas por GPI/genética , Regulação Neoplásica da Expressão Gênica/genética , Xenoenxertos , Humanos , Camundongos , Mitocôndrias/genética , Terapia de Alvo Molecular , Proteínas Proto-Oncogênicas p21(ras)/genética , Linfócitos T/patologia
4.
Oncotarget ; 9(89): 35875-35890, 2018 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-30542505

RESUMO

Double-hit (DH) or double-expresser (DE) lymphomas are high-grade diffuse large B-cell lymphomas (DLBCL) that are mostly incurable with standard chemo-immunotherapy due to treatment resistance. The generation of drug-induced aneuploid/polyploid (DIAP) cells is a common effect of anti-DLBCL therapies (e.g. vincristine, doxorubicin). DIAP cells are thought to be responsible for treatment resistance, as they are capable of re-entering the cell cycle during off-therapy periods. Previously we have shown that combination of alisertib plus ibrutinib plus rituximab can partially abrogate DIAP cells and induce cell death. Here, we provide evidence that DIAP cells can re-enter the cell cycle and escape cell death during anti-DLBCL treatment. We also discuss MYC/BCL2 mediated molecular mechanism that underlie treatment resistance. We isolated aneuploid/polyploid populations of DH/DE-DLBCL cells after treatment with the aurora kinase (AK) inhibitor alisertib. Time-lapse microscopy of single polyploid cells revealed that following drug removal, a subset of these DIAP cells divide and proliferate by reductive cell divisions, including multipolar mitosis, meiosis-like nuclear fission and budding. Genomic, proteomic, and kinomic profiling demonstrated that alisertib-induced aneuploid/polyploid cells up-regulate DNA damage, DNA replication and immune evasion pathways. In addition, we identified amplified receptor tyrosine kinase and T-cell receptor signaling, as well as MYC-mediated dysregulation of the spindle assembly checkpoints RanGAP1, TPX2 and KPNA2. We infer that these factors contribute to treatment resistance of DIAP cells. These findings provide opportunities to develop novel DH/DE-DLBCL therapies, specifically targeting DIAP cells. KEY POINTS: ● MYC mediated upregulation of TPX2, KPNA2 and RanGAP1 dysregulate the spindle assembly checkpoint in drug-induced polyploid cells.● Drug-induced polyploid cells re-enter the cell cycle via multipolar mitosis, fission or budding, a mechanism of disease relapse.

5.
Oncotarget ; 8(59): 100326-100338, 2017 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-29245981

RESUMO

Peripheral T-cell non-Hodgkin lymphoma (PTCL) are heterogeneous, rare, and aggressive diseases mostly incurable with current cell cycle therapies. Aurora kinases (AKs) are key regulators of mitosis that drive PTCL proliferation. Alisertib (AK inhibitor) has a response rate ∼30% in relapsed and refractory PTCL (SWOG1108). Since PTCL are derived from CD4+/CD8+ cells, we hypothesized that Program Death Ligand-1 (PD-L1) expression is essential for uncontrolled proliferation. Combination of alisertib with PI3Kα (MLN1117) or pan-PI3K inhibition (PF-04691502) or vincristine (VCR) was highly synergistic in PTCL cells. Expression of PD-L1 relative to PD-1 is high in PTCL biopsies (∼9-fold higher) and cell lines. Combination of alisertib with pan-PI3K inhibition or VCR significantly reduced PD-L1, NF-κB expression and inhibited phosphorylation of AKT, ERK1/2 and AK with enhanced apoptosis. In a SCID PTCL xenograft mouse model, alisertib displayed high synergism with MLN1117. In a syngeneic PTCL mouse xenograft model alisertib demonstrated tumor growth inhibition (TGI) ∼30%, whilst anti-PD-L1 therapy alone was ineffective. Alisertib + anti-PD-L1 resulted in TGI >90% indicative of a synthetic lethal interaction. PF-04691502 + alisertib + anti-PD-L1 + VCR resulted in TGI 100%. Overall, mice tolerated the treatments well. Co-targeting AK, PI3K and PD-L1 is a rational and novel therapeutic strategy for PTCL.

6.
Mol Cancer Ther ; 16(10): 2083-2093, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28615297

RESUMO

Double hit (DH) or double expressor (DE) diffuse large B-cell lymphomas (DLBCL) are aggressive non-Hodgkin's lymphomas (NHL) with translocations and/or overexpressions of MYC and BCL-2, which are difficult to treat. Aurora kinase (AK) inhibition with alisertib in DH/DE-DLBCL induces cell death in ∼30%, while ∼70% are aneuploid and senescent cells (AASC), a mitotic escape mechanism contributing to drug resistance. These AASCs elaborated a high metabolic rate by increased AKT/mTOR and ERK/MAPK activity via BTK signaling through the chronic active B-cell receptor (BCR) pathway. Combinations of alisertib + ibrutinib or alisertib + ibrutinib + rituximab significantly reduced AASCs with enhanced intrinsic cell death. Inhibition of AK + BTK reduced phosphorylation of AKT/mTOR and ERK-1/2, upregulated phospho-H2A-X and Chk-2 (DNA damage), reduced Bcl-6, and decreased Bcl-2 and Bcl-xL and induced apoptosis by PARP cleavage. In a DE-DLBCL SCID mouse xenograft model, ibrutinib alone was inactive, while alisertib + ibrutinib was additive with a tumor growth inhibition (TGI) rate of ∼25%. However, TGI for ibrutinib + rituximab was ∼50% to 60%. In contrast, triple therapy showed a TGI rate of >90%. Kaplan-Meier survival analysis showed that 67% of mice were alive at day 89 with triple therapy versus 20% with ibrutinib + rituximab. All treatments were well tolerated with no changes in body weights. A novel triple therapy consisting of alisertib + ibrutinib + rituximab inhibits AASCs induced by AK inhibition in DH/DE-DLBCL leading to a significant antiproliferative signal, enhanced intrinsic apoptosis and may be of therapeutic potential in these lymphomas. Mol Cancer Ther; 16(10); 2083-93. ©2017 AACR.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Aurora Quinase A/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Adenina/análogos & derivados , Aneuploidia , Animais , Apoptose/efeitos dos fármacos , Azepinas/administração & dosagem , Linhagem Celular Tumoral , Senescência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Piperidinas , Inibidores de Proteínas Quinases/administração & dosagem , Pirazóis/administração & dosagem , Pirimidinas/administração & dosagem , Rituximab/administração & dosagem , Serina-Treonina Quinases TOR/genética , Ensaios Antitumorais Modelo de Xenoenxerto
7.
DNA Repair (Amst) ; 43: 98-106, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27130816

RESUMO

Non-Homologous End-Joining (NHEJ) is the predominant pathway for the repair of DNA double strand breaks (DSBs) in human cells. The NHEJ pathway is frequently upregulated in several solid cancers as a compensatory mechanism for a separate DSB repair defect or for innate genomic instability, making this pathway a powerful target for synthetic lethality approaches. In addition, NHEJ reduces the efficacy of cancer treatment modalities which rely on the introduction of DSBs, like radiation therapy or genotoxic chemotherapy. Consequently, inhibition of the NHEJ pathway can modulate a radiation- or chemo-refractory disease presentation. The Ku70/80 heterodimer protein plays a pivotal role in the NHEJ process. It possesses a ring-shaped structure with high affinity for DSBs and serves as the first responder and central scaffold around which the rest of the repair complex is assembled. Because of this central position, the Ku70/80 dimer is a logical target for the disruption of the entire NHEJ pathway. Surprisingly, specific inhibitors of the Ku70/80 heterodimer are currently not available. We here describe an in silico, pocket-based drug discovery methodology utilizing the crystal structure of the Ku70/80 heterodimer. We identified a novel putative small molecule binding pocket and selected several potential inhibitors by computational screening. Subsequent biological screening resulted in the first identification of a compound with confirmed Ku-inhibitory activity in the low micro-molar range, capable of disrupting the binding of Ku70/80 to DNA substrates and impairing Ku-dependent activation of another NHEJ factor, the DNA-PKCS kinase. Importantly, this compound synergistically sensitized human cell lines to radiation treatment, indicating a clear potential to diminish DSB repair. The chemical scaffold we here describe can be utilized as a lead-generating platform for the design and development of a novel class of anti-cancer agents.


Assuntos
Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Proteína Quinase Ativada por DNA/antagonistas & inibidores , DNA/genética , Autoantígeno Ku/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Pirimidinas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Cristalografia por Raios X , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Proteína Quinase Ativada por DNA/química , Proteína Quinase Ativada por DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Raios gama , Expressão Gênica , Células HeLa , Humanos , Autoantígeno Ku/química , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Simulação de Acoplamento Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Domínios Proteicos , Multimerização Proteica , Estrutura Secundária de Proteína , Pirimidinas/síntese química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química
8.
Front Biosci (Landmark Ed) ; 21(3): 514-27, 2016 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-26709791

RESUMO

Non-homologous end-joining (NHEJ) is an essential DNA double strand break repair pathway during all cell cycle stages. Deficiency in NHEJ factors can lead to accumulation of unrepaired DNA breaks or faulty DNA repair, which may ultimately result in cell death, senescence or carcinogenesis. The Ku70/80 heterodimer is a key-player in the NHEJ pathway and binds to DNA termini with high affinity, where it helps to protect DNA ends from degradation and to recruit other NHEJ factors required for repair. The mechanism of Ku70/80 detachment from the DNA helix after completion of DNA repair is incompletely understood. Some data suggest that certain DNA repair factors are ubiquitylated and targeted for proteasomal degradation after repair. Recent studies suggest that Ku80 is conjugated to lysine48-linked ubiquitin chains by the Skp1-Cullin-F-box (SCF) complex and/or the RING finger protein 8 (RNF8) ubiquitin-protein ligases, followed by rapid proteasomal degradation. In this review we address the structure and function of the Ku70/80 heterodimer and how ubiquitylation may affect the release of Ku70/80 from chromatin and its subsequent degradation via the ubiquitin-proteasome system.


Assuntos
Antígenos Nucleares/fisiologia , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/fisiologia , Dano ao DNA , Reparo do DNA , Autoantígeno Ku , Ubiquitina-Proteína Ligases/metabolismo
9.
Mol Cell Biol ; 29(5): 1134-42, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19103741

RESUMO

Repair of DNA double-strand breaks (DSBs) is predominantly mediated by nonhomologous end joining (NHEJ) in mammalian cells. NHEJ requires binding of the Ku70-Ku80 heterodimer (Ku70/80) to the DNA ends and subsequent recruitment of the DNA-dependent protein kinase catalytic subunit (DNA-PK(CS)) and the XRCC4/ligase IV complex. Activation of the DNA-PK(CS) serine/threonine kinase requires an interaction with Ku70/80 and is essential for NHEJ-mediated DSB repair. In contrast to previous models, we found that the carboxy terminus of Ku80 is not absolutely required for the recruitment and activation of DNA-PK(CS) at DSBs, although cells that harbored a carboxy-terminal deletion in the Ku80 gene were sensitive to ionizing radiation and showed reduced end-joining capacity. More detailed analysis of this repair defect showed that DNA-PK(CS) autophosphorylation at Thr2647 was diminished, while Ser2056 was phosphorylated to normal levels. This resulted in severely reduced levels of Artemis nuclease activity in vivo and in vitro. We therefore conclude that the Ku80 carboxy terminus is important to support DNA-PK(CS) autophosphorylation at specific sites, which facilitates DNA end processing by the Artemis endonuclease and the subsequent joining reaction.


Assuntos
Antígenos Nucleares/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas Nucleares/metabolismo , Deleção de Sequência , Animais , Antígenos Nucleares/genética , Domínio Catalítico , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Enzimas Reparadoras do DNA/metabolismo , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/genética , Endonucleases , Humanos , Autoantígeno Ku , Fragmentos de Peptídeos , Fosforilação , Radiação Ionizante
10.
Cell Res ; 18(1): 114-24, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18166980

RESUMO

DNA double-strand breaks (DSBs) are introduced in cells by ionizing radiation and reactive oxygen species. In addition, they are commonly generated during V(D)J recombination, an essential aspect of the developing immune system. Failure to effectively repair these DSBs can result in chromosome breakage, cell death, onset of cancer, and defects in the immune system of higher vertebrates. Fortunately, all mammalian cells possess two enzymatic pathways that mediate the repair of DSBs: homologous recombination and non-homologous end-joining (NHEJ). The NHEJ process utilizes enzymes that capture both ends of the broken DNA molecule, bring them together in a synaptic DNA-protein complex, and finally repair the DNA break. In this review, all the known enzymes that play a role in the NHEJ process are discussed and a working model for the co-operation of these enzymes during DSB repair is presented.


Assuntos
Reparo do DNA/fisiologia , Animais , Quebras de DNA de Cadeia Dupla , Rearranjo Gênico/imunologia , Rearranjo Gênico/fisiologia , Humanos , Modelos Biológicos , Recombinação Genética/imunologia , Éxons VDJ/genética
11.
EMBO Rep ; 9(1): 91-6, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18064046

RESUMO

XRCC4-like factor (XLF)--also known as Cernunnos--has recently been shown to be involved in non-homologous end-joining (NHEJ), which is the main pathway for the repair of DNA double-strand breaks (DSBs) in mammalian cells. XLF is likely to enhance NHEJ by stimulating XRCC4-ligase IV-mediated joining of DSBs. Here, we report mechanistic details of XLF recruitment to DSBs. Live cell imaging combined with laser micro-irradiation showed that XLF is an early responder to DSBs and that Ku is essential for XLF recruitment to DSBs. Biochemical analysis showed that Ku-XLF interaction occurs on DNA and that Ku stimulates XLF binding to DNA. Unexpectedly, XRCC4 is dispensable for XLF recruitment to DSBs, although photobleaching analysis showed that XRCC4 stabilizes the binding of XLF to DSBs. Our observations showed the direct involvement of XLF in the dynamic assembly of the NHEJ machinery and provide mechanistic insights into DSB recognition.


Assuntos
Antígenos Nucleares/metabolismo , Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Linhagem Celular , DNA/metabolismo , Humanos , Autoantígeno Ku , Lasers , Ligação Proteica , Termodinâmica
12.
J Cell Biol ; 179(2): 183-6, 2007 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-17938249

RESUMO

The DNA-dependent protein kinase (DNA-PK) is one of the central enzymes involved in DNA double-strand break (DSB) repair. It facilitates proper alignment of the two ends of the broken DNA molecule and coordinates access of other factors to the repair complex. We discuss the latest findings on DNA-PK phosphorylation and offer a working model for the regulation of DNA-PK during DSB repair.


Assuntos
Proteína Quinase Ativada por DNA/metabolismo , Recombinação Genética , Animais , Domínio Catalítico , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteína Quinase Ativada por DNA/química , Humanos , Fosforilação
13.
J Cell Biol ; 177(2): 219-29, 2007 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-17438073

RESUMO

The DNA-dependent protein kinase catalytic subunit (DNA-PK(CS)) plays an important role during the repair of DNA double-strand breaks (DSBs). It is recruited to DNA ends in the early stages of the nonhomologous end-joining (NHEJ) process, which mediates DSB repair. To study DNA-PK(CS) recruitment in vivo, we used a laser system to introduce DSBs in a specified region of the cell nucleus. We show that DNA-PK(CS) accumulates at DSB sites in a Ku80-dependent manner, and that neither the kinase activity nor the phosphorylation status of DNA-PK(CS) influences its initial accumulation. However, impairment of both of these functions results in deficient DSB repair and the maintained presence of DNA-PK(CS) at unrepaired DSBs. The use of photobleaching techniques allowed us to determine that the kinase activity and phosphorylation status of DNA-PK(CS) influence the stability of its binding to DNA ends. We suggest a model in which DNA-PK(CS) phosphorylation/autophosphorylation facilitates NHEJ by destabilizing the interaction of DNA-PK(CS) with the DNA ends.


Assuntos
Domínio Catalítico , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteína Quinase Ativada por DNA/metabolismo , Animais , Antígenos Nucleares/metabolismo , Células CHO , Cricetinae , Cricetulus , DNA/metabolismo , Proteína Quinase Ativada por DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Autoantígeno Ku , Lasers , Fosforilação , Fotodegradação
14.
DNA Repair (Amst) ; 3(11): 1425-35, 2004 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-15380098

RESUMO

Repair of DNA double-strand breaks (DSBs) by non-homologous end-joining (NHEJ) is required for resistance to genotoxic agents, such as ionizing radiation, but also for proper development of the vertebrate immune system. Much progress has been made in identifying the factors that are involved in this repair pathway. We are now entering the phase in which we begin to understand basic concepts of the reaction mechanism and regulation of non-homologous end-joining. This review concentrates on novel insights into damage recognition and subsequent tethering, processing and joining of DNA ends.


Assuntos
Reparo do DNA/fisiologia , Animais , DNA/química , DNA/genética , DNA/metabolismo , Dano ao DNA , DNA Ligase Dependente de ATP , DNA Ligases/metabolismo , Reparo do DNA/genética , Reparo do DNA/imunologia , Enzimas Reparadoras do DNA/metabolismo , Proteína Quinase Ativada por DNA , Proteínas de Ligação a DNA/metabolismo , Genes de Imunoglobulinas , Genes Codificadores dos Receptores de Linfócitos T , Humanos , Modelos Biológicos , Proteínas Nucleares , Proteínas Serina-Treonina Quinases/metabolismo
15.
Nucleic Acids Res ; 31(24): 7238-46, 2003 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-14654699

RESUMO

DNA dependent protein kinase (DNA-PK) plays a central role in the non-homologous end-joining pathway of DNA double strand break repair. Its catalytic subunit (DNA-PK(CS)) functions as a serine/threonine protein kinase. We show that DNA-PK forms a stable complex at DNA termini that blocks the action of exonucleases and ligases. The DNA termini become accessible after autophosphorylation of DNA-PK(CS), which we demonstrate to require synapsis of DNA ends. Interestingly, the presence of DNA-PK prevents ligation of the two synapsed termini, but allows ligation to another DNA molecule. This alteration of the ligation route is independent of the type of ligase that we used, indicating that the intrinsic architecture of the DNA-PK complex itself is not able to support ligation of the synapsed DNA termini. We present a working model in which DNA-PK creates a stable molecular bridge between two DNA ends that is remodeled after DNA-PK autophosphorylation in such a way that the extreme termini become accessible without disrupting synapsis. We infer that joining of synapsed DNA termini would require an additional protein factor.


Assuntos
DNA/química , DNA/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Recombinação Genética , Trifosfato de Adenosina/metabolismo , Catálise , Pegada de DNA , DNA Ligases/metabolismo , Proteína Quinase Ativada por DNA , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/enzimologia , Humanos , Modelos Biológicos , Proteínas Nucleares , Fosforilação , Ligação Proteica
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