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1.
Cell ; 136(3): 420-34, 2009 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-19203578

RESUMO

The biological response to DNA double-strand breaks acts to preserve genome integrity. Individuals bearing inactivating mutations in components of this response exhibit clinical symptoms that include cellular radiosensitivity, immunodeficiency, and cancer predisposition. The archetype for such disorders is Ataxia-Telangiectasia caused by biallelic mutation in ATM, a central component of the DNA damage response. Here, we report that the ubiquitin ligase RNF168 is mutated in the RIDDLE syndrome, a recently discovered immunodeficiency and radiosensitivity disorder. We show that RNF168 is recruited to sites of DNA damage by binding to ubiquitylated histone H2A. RNF168 acts with UBC13 to amplify the RNF8-dependent histone ubiquitylation by targeting H2A-type histones and by promoting the formation of lysine 63-linked ubiquitin conjugates. These RNF168-dependent chromatin modifications orchestrate the accumulation of 53BP1 and BRCA1 to DNA lesions, and their loss is the likely cause of the cellular and developmental phenotypes associated with RIDDLE syndrome.


Assuntos
Dano ao DNA , Síndromes de Imunodeficiência/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo , Linhagem Celular , Histonas/metabolismo , Humanos , Síndromes de Imunodeficiência/genética , Tolerância a Radiação , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
2.
Mol Cell ; 59(3): 462-77, 2015 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-26166705

RESUMO

Recognition and repair of damaged replication forks are essential to maintain genome stability and are coordinated by the combined action of the Fanconi anemia and homologous recombination pathways. These pathways are vital to protect stalled replication forks from uncontrolled nucleolytic activity, which otherwise causes irreparable genomic damage. Here, we identify BOD1L as a component of this fork protection pathway, which safeguards genome stability after replication stress. Loss of BOD1L confers exquisite cellular sensitivity to replication stress and uncontrolled resection of damaged replication forks, due to a failure to stabilize RAD51 at these forks. Blocking DNA2-dependent resection, or downregulation of the helicases BLM and FBH1, suppresses both catastrophic fork processing and the accumulation of chromosomal damage in BOD1L-deficient cells. Thus, our work implicates BOD1L as a critical regulator of genome integrity that restrains nucleolytic degradation of damaged replication forks.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Linhagem Celular , Sobrevivência Celular , Dano ao DNA , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Genoma Humano , Instabilidade Genômica , Células HeLa , Humanos , RecQ Helicases/metabolismo
3.
Am J Hum Genet ; 96(3): 412-24, 2015 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-25728776

RESUMO

Non-homologous end joining (NHEJ) is a key cellular process ensuring genome integrity. Mutations in several components of the NHEJ pathway have been identified, often associated with severe combined immunodeficiency (SCID), consistent with the requirement for NHEJ during V(D)J recombination to ensure diversity of the adaptive immune system. In contrast, we have recently found that biallelic mutations in LIG4 are a common cause of microcephalic primordial dwarfism (MPD), a phenotype characterized by prenatal-onset extreme global growth failure. Here we provide definitive molecular genetic evidence supported by biochemical, cellular, and immunological data for mutations in XRCC4, encoding the obligate binding partner of LIG4, causing MPD. We report the identification of biallelic mutations in XRCC4 in five families. Biochemical and cellular studies demonstrate that these alterations substantially decrease XRCC4 protein levels leading to reduced cellular ligase IV activity. Consequently, NHEJ-dependent repair of ionizing-radiation-induced DNA double-strand breaks is compromised in XRCC4 cells. Similarly, immunoglobulin junctional diversification is impaired in cells. However, immunoglobulin levels are normal, and individuals lack overt signs of immunodeficiency. Additionally, in contrast to individuals with LIG4 mutations, pancytopenia leading to bone marrow failure has not been observed. Hence, alterations that alter different NHEJ proteins give rise to a phenotypic spectrum, from SCID to extreme growth failure, with deficiencies in certain key components of this repair pathway predominantly exhibiting growth deficits, reflecting differential developmental requirements for NHEJ proteins to support growth and immune maturation.


Assuntos
Proteínas de Ligação a DNA/genética , Nanismo Hipofisário/genética , Nanismo/genética , Microcefalia/genética , Mutação , Alelos , Sequência de Aminoácidos , Criança , Pré-Escolar , Quebras de DNA de Cadeia Dupla , DNA Ligase Dependente de ATP , DNA Ligases/genética , DNA Ligases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Campo Pulsado , Exoma , Fácies , Feminino , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Fenótipo , Conformação Proteica , Imunodeficiência Combinada Severa/genética
4.
EMBO J ; 32(12): 1778-92, 2013 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-23708797

RESUMO

Limiting the levels of homologous recombination (HR) that occur at sites of DNA damage is a major role of BLM helicase. However, very little is known about the mechanisms dictating its relocalization to these sites. Here, we demonstrate that the ubiquitin/SUMO-dependent DNA damage response (UbS-DDR), controlled by the E3 ligases RNF8/RNF168, triggers BLM recruitment to sites of replication fork stalling via ubiquitylation in the N-terminal region of BLM and subsequent BLM binding to the ubiquitin-interacting motifs of RAP80. Furthermore, we show that this mechanism of BLM relocalization is essential for BLM's ability to suppress excessive/uncontrolled HR at stalled replication forks. Unexpectedly, we also uncovered a requirement for RNF8-dependent ubiquitylation of BLM and PML for maintaining the integrity of PML-associated nuclear bodies and as a consequence the localization of BLM to these structures. Lastly, we identified a novel role for RAP80 in preventing proteasomal degradation of BLM in unstressed cells. Taken together, these data highlight an important biochemical link between the UbS-DDR and BLM-dependent pathways involved in maintaining genome stability.


Assuntos
Dano ao DNA , Instabilidade Genômica/fisiologia , Recombinação Homóloga/fisiologia , Proteólise , RecQ Helicases/metabolismo , Ubiquitinação/fisiologia , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , RecQ Helicases/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
5.
J Biol Chem ; 284(49): 33939-48, 2009 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-19826003

RESUMO

Human mediator of DNA damage checkpoint 1 (hMDC1) is an essential component of the cellular response to DNA double strand breaks. Recently, hMDC1 has been shown to associate with a subunit of the anaphase-promoting complex/cyclosome (APC/C) (Coster, G., Hayouka, Z., Argaman, L., Strauss, C., Friedler, A., Brandeis, M., and Goldberg, M. (2007) J. Biol. Chem. 282, 32053-32064), a key regulator of mitosis, suggesting a possible role for hMDC1 in controlling normal cell cycle progression. Here, we extend this work to show that hMDC1 regulates normal metaphase-to-anaphase transition through its ability to bind directly to the APC/C and modulate its E3 ubiquitin ligase activity. In support of a role for hMDC1 in controlling mitotic progression, depletion of hMDC1 by small interfering RNA results in a metaphase arrest that appears to be independent of both BubR1-dependent signaling pathways and ATM/ATR activation. Mitotic cells lacking hMDC1 exhibit markedly reduced levels of APC/C activity characterized by reduced levels of Cdc20, and a failure of Cdc20 to bind the APC/C and CREB-binding protein. We suggest therefore that hMDC1 functionally regulates the normal metaphase-to-anaphase transition by modulating the Cdc20-dependent activation of the APC/C.


Assuntos
Mitose , Proteínas Nucleares/fisiologia , Transativadores/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Anáfase , Ciclossomo-Complexo Promotor de Anáfase , Proteínas Cdc20 , Proteínas de Ciclo Celular/metabolismo , Células HeLa , Humanos , Immunoblotting/métodos , Metáfase , Microscopia de Fluorescência/métodos , Modelos Biológicos , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Complexos Ubiquitina-Proteína Ligase/química , Ubiquitina-Proteína Ligases/química
6.
J Cell Biol ; 181(2): 227-40, 2008 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-18411308

RESUMO

The MRE11-RAD50-Nijmegen breakage syndrome 1 (NBS1 [MRN]) complex accumulates at sites of DNA double-strand breaks (DSBs) in microscopically discernible nuclear foci. Focus formation by the MRN complex is dependent on MDC1, a large nuclear protein that directly interacts with phosphorylated H2AX. In this study, we identified a region in MDC1 that is essential for the focal accumulation of the MRN complex at sites of DNA damage. This region contains multiple conserved acidic sequence motifs that are constitutively phosphorylated in vivo. We show that these motifs are efficiently phosphorylated by caseine kinase 2 (CK2) in vitro and directly interact with the N-terminal forkhead-associated domain of NBS1 in a phosphorylation-dependent manner. Mutation of these conserved motifs in MDC1 or depletion of CK2 by small interfering RNA disrupts the interaction between MDC1 and NBS1 and abrogates accumulation of the MRN complex at sites of DNA DSBs in vivo. Thus, our data reveal the mechanism by which MDC1 physically couples the MRN complex to damaged chromatin.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Dano ao DNA , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Hidrolases Anidrido Ácido , Proteínas Adaptadoras de Transdução de Sinal , Animais , Caseína Quinase II/metabolismo , DNA Complementar , Vetores Genéticos , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína Homóloga a MRE11 , Camundongos , Fosforilação , RNA Interferente Pequeno/genética , Transfecção
7.
Proc Natl Acad Sci U S A ; 104(43): 16910-5, 2007 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-17940005

RESUMO

Cellular DNA double-strand break-repair pathways have evolved to protect the integrity of the genome from a continual barrage of potentially detrimental insults. Inherited mutations in genes that control this process result in an inability to properly repair DNA damage, ultimately leading to developmental defects and also cancer predisposition. Here, we describe a patient with a previously undescribed syndrome, which we have termed RIDDLE syndrome (radiosensitivity, immunodeficiency, dysmorphic features and learning difficulties), whose cells lack an ability to recruit 53BP1 to sites of DNA double-strand breaks. As a consequence, cells derived from this patient exhibit a hypersensitivity to ionizing radiation, cell cycle checkpoint abnormalities, and impaired end-joining in the recombined switch regions. Sequencing of TP53BP1 and other genes known to regulate ionizing radiation-induced 53BP1 foci formation in this patient failed to detect any mutations. Therefore, these data indicate the existence of a DNA double-strand break-repair protein that functions upstream of 53BP1 and contributes to the normal development of the human immune system.


Assuntos
Dano ao DNA , Síndromes de Imunodeficiência/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/efeitos da radiação , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática/efeitos da radiação , Fibroblastos/patologia , Fibroblastos/efeitos da radiação , Humanos , Switching de Imunoglobulina/efeitos da radiação , Síndromes de Imunodeficiência/patologia , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico/efeitos da radiação , Tolerância a Radiação , Radiação Ionizante , Recombinação Genética/efeitos da radiação , Transdução de Sinais/efeitos da radiação , Hipermutação Somática de Imunoglobulina/genética , Hipermutação Somática de Imunoglobulina/efeitos da radiação , Transativadores/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53
8.
J Ind Microbiol Biotechnol ; 34(4): 289-99, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17205350

RESUMO

Methylomonas sp. strain 16a is an obligate methanotrophic bacterium that uses methane or methanol as the sole carbon source. An effort was made to engineer this organism for astaxanthin production. Upon expressing the canthaxanthin gene cluster under the control of the native hps promoter in the chromosome, canthaxanthin was produced as the main carotenoid. Further conversion to astaxanthin was carried out by expressing different combinations of crtW and crtZ genes encoding the beta-carotenoid ketolase and hydroxylase. The carotenoid intermediate profile was influenced by the copy number of these two genes under the control of the hps promoter. Expression of two copies of crtZ and one copy of crtW led to the accumulation of a large amount of the mono-ketolated product adonixanthin. On the other hand, expression of two copies of crtW and one copy of crtZ resulted in the presence of non-hydroxylated carotenoid canthaxanthin and the mono-hydroxylated adonirubin. Production of astaxanthin as the predominant carotenoid was obtained in a strain containing two complete sets of carotenoid biosynthetic genes. This strain had an astaxanthin titer ranging from 1 to 2.4 mg g(-1) of dry cell biomass depending on the growth conditions. More than 90% of the total carotenoid was astaxanthin, of which the majority was in the form of E-isomer. This result indicates that it is possible to produce astaxanthin with desirable properties in methanotrophs through genetic engineering.


Assuntos
Methylomonas/genética , Methylomonas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Cantaxantina/química , Cantaxantina/metabolismo , Carotenoides/química , Carotenoides/metabolismo , Cromatografia Líquida de Alta Pressão , Fermentação , Metano/metabolismo , Modelos Biológicos , Estrutura Molecular , Xantofilas/biossíntese , Xantofilas/química
9.
Biotechnol Bioeng ; 77(3): 340-51, 2002 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-11753943

RESUMO

A microbial oxidation process for the production of p-hydroxybenzoate (HBA) from toluene is reported. The oxidation reaction was studied in fed-batch fermentations using a recombinant Pseudomonas putida grown on glutamate as the sole carbon and energy source with salicylate and IPTG induction of tmoABCDE, and pchCF and phbz pathway genes, respectively. An average volumetric HBA productivity of 13.4 mg HBA x L(-1) x h(-1) was obtained under rapid growth conditions (glutamate excess), giving an HBA titer of 132 mg x L(-1) after 9.8 h of fermentation. This corresponded to an average specific HBA productivity of 7.2 microg HBA (mg total protein)(-1) x h(-1). In contrast, maximum HBA titers of 35 mg HBA x L(-1) were achieved in 27 h in comparative studies employing glutumate limited fed-batch cultures. A specific productivity of 4.1 microg HBA (mg total protein)(-1) x h(-1) and volumetric productivity of 1.3 mg HBA x L(-1) x h(-1) were calculated for the growth-rate restricted cultures. The differences in HBA production between the two cultures could be correlated to the levels of specific toluene-4-monooxygenase (T4MO) polypeptides. T4MO catalyzes the rate-limiting step in the pathway. Using experimental data, the half-life value of TmoA was calculated to be approximately 28 h. Assuming linear, monomolecular decay of TmoA, a specific degradation constant of 0.025 x h(-1) was calculated, which placed the stability of recombinant TmoA in the range of relatively stable proteins, even in the absence of co-expression of tmoF, the terminal oxidoreductase subunit of T4MO.


Assuntos
Microbiologia Industrial/métodos , Parabenos/metabolismo , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Tolueno/metabolismo , Divisão Celular/genética , Fermentação , Engenharia Genética/métodos , Modelos Biológicos
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