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1.
Nat Commun ; 12(1): 6078, 2021 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-34667177

RESUMO

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay.


Assuntos
Neurônios/metabolismo , RNA Polimerase II/química , RNA Polimerase II/metabolismo , RNA , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Camundongos Knockout , Neurônios/química , Fosforilação , Domínios Proteicos , RNA/química , RNA/genética , RNA/metabolismo , RNA Polimerase II/genética , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , Fatores de Transcrição/genética , Transcrição Genética
2.
Nucleic Acids Res ; 48(21): e122, 2020 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-33053171

RESUMO

Protein-protein interactions are essential to ensure timely and precise recruitment of chromatin remodellers and repair factors to DNA damage sites. Conventional analyses of protein-protein interactions at a population level may mask the complexity of interaction dynamics, highlighting the need for a method that enables quantification of DNA damage-dependent interactions at a single-cell level. To this end, we integrated a pulsed UV laser on a confocal fluorescence lifetime imaging (FLIM) microscope to induce localized DNA damage. To quantify protein-protein interactions in live cells, we measured Förster resonance energy transfer (FRET) between mEGFP- and mCherry-tagged proteins, based on the fluorescence lifetime reduction of the mEGFP donor protein. The UV-FLIM-FRET system offers a unique combination of real-time and single-cell quantification of DNA damage-dependent interactions, and can distinguish between direct protein-protein interactions, as opposed to those mediated by chromatin proximity. Using the UV-FLIM-FRET system, we show the dynamic changes in the interaction between poly(ADP-ribose) polymerase 1, amplified in liver cancer 1, X-ray repair cross-complementing protein 1 and tripartite motif containing 33 after DNA damage. This new set-up complements the toolset for studying DNA damage response by providing single-cell quantitative and dynamic information about protein-protein interactions at DNA damage sites.


Assuntos
Osteoblastos/efeitos da radiação , Poli(ADP-Ribose) Polimerase-1/genética , Mapeamento de Interação de Proteínas/métodos , Fatores de Transcrição/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Cromatina/efeitos da radiação , Dano ao DNA , Transferência Ressonante de Energia de Fluorescência , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lasers , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Imagem Óptica , Osteoblastos/citologia , Osteoblastos/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Ligação Proteica , Transdução de Sinais , Análise de Célula Única , Fatores de Transcrição/metabolismo , Raios Ultravioleta , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo
3.
Curr Biol ; 30(15): 3045-3056.e7, 2020 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-32589908

RESUMO

Centrioles are highly elaborate microtubule-based structures responsible for the formation of centrosomes and cilia. Despite considerable variation across species and tissues within any given tissue, their size is essentially constant [1, 2]. While the diameter of the centriole cylinder is set by the dimensions of the inner scaffolding structure of the cartwheel [3], how centriole length is set so precisely and stably maintained over many cell divisions is not well understood. Cep97 and CP110 are conserved proteins that localize to the distal end of centrioles and have been reported to limit centriole elongation in vertebrates [4, 5]. Here, we examine Cep97 function in Drosophila melanogaster. We show that Cep97 is essential for formation of full-length centrioles in multiple tissues of the fly. We further identify the microtubule deacetylase Sirt2 as a Cep97 interactor. Deletion of Sirt2 likewise affects centriole size. Interestingly, so does deletion of the acetylase Atat1, indicating that loss of stabilizing acetyl marks impairs centriole integrity. Cep97 and CP110 were originally identified as inhibitors of cilia formation in vertebrate cultured cells [6], and loss of CP110 is a widely used marker of basal body maturation. In contrast, in Drosophila, Cep97 appears to be only transiently removed from basal bodies and loss of Cep97 strongly impairs ciliogenesis. Collectively, our results support a model whereby Cep97 functions as part of a protective cap that acts together with the microtubule acetylation machinery to maintain centriole stability, essential for proper function in cilium biogenesis.


Assuntos
Centríolos/fisiologia , Centrossomo , Cílios , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Proteínas Associadas aos Microtúbulos/fisiologia , Morfogênese/genética , Animais , Corpos Basais/metabolismo , Células Cultivadas , Centrossomo/metabolismo , Cílios/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Morfogênese/fisiologia
4.
Science ; 368(6486): 30-31, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32241937
5.
Genes Dev ; 34(5-6): 360-394, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32029455

RESUMO

Oxidative and replication stress underlie genomic instability of cancer cells. Amplifying genomic instability through radiotherapy and chemotherapy has been a powerful but nonselective means of killing cancer cells. Precision medicine has revolutionized cancer therapy by putting forth the concept of selective targeting of cancer cells. Poly(ADP-ribose) polymerase (PARP) inhibitors represent a successful example of precision medicine as the first drugs targeting DNA damage response to have entered the clinic. PARP inhibitors act through synthetic lethality with mutations in DNA repair genes and were approved for the treatment of BRCA mutated ovarian and breast cancer. PARP inhibitors destabilize replication forks through PARP DNA entrapment and induce cell death through replication stress-induced mitotic catastrophe. Inhibitors of poly(ADP-ribose) glycohydrolase (PARG) exploit and exacerbate replication deficiencies of cancer cells and may complement PARP inhibitors in targeting a broad range of cancer types with different sources of genomic instability. Here I provide an overview of the molecular mechanisms and cellular consequences of PARP and PARG inhibition. I highlight clinical performance of four PARP inhibitors used in cancer therapy (olaparib, rucaparib, niraparib, and talazoparib) and discuss the predictive biomarkers of inhibitor sensitivity, mechanisms of resistance as well as the means of overcoming them through combination therapy.


Assuntos
Antineoplásicos/uso terapêutico , Glicosídeo Hidrolases/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Instabilidade Genômica , Glicosídeo Hidrolases/metabolismo , Humanos , Neoplasias/enzimologia , Poli(ADP-Ribose) Polimerases/metabolismo
6.
J Cell Sci ; 132(21)2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31604796

RESUMO

Sirtuin 2 (SIRT2) is an NAD-dependent sirtuin deacetylase that regulates microtubule and chromatin dynamics, gene expression and cell cycle progression, as well as nuclear envelope reassembly. Recent proteomic analyses have identified Golgi proteins as SIRT2 interactors, indicating that SIRT2 may also play a role in Golgi structure formation. Here, we show that SIRT2 depletion causes Golgi fragmentation and impairs Golgi reassembly at the end of mitosis. SIRT2 interacts with the Golgi reassembly stacking protein GRASP55 (also known as GORASP2) in mitosis when GRASP55 is highly acetylated on K50. Expression of wild-type and the K50R acetylation-deficient mutant of GRASP55, but not the K50Q acetylation-mimetic mutant, in GRASP55 and GRASP65 (also known as GORASP1) double-knockout cells, rescued the Golgi structure and post-mitotic Golgi reassembly. Acetylation-deficient GRASP55 exhibited a higher self-interaction efficiency, a property required for Golgi structure formation. These results demonstrate that SIRT2 regulates Golgi structure by modulating GRASP55 acetylation levels.


Assuntos
Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi/metabolismo , Microtúbulos/metabolismo , Sirtuína 2/metabolismo , Humanos , Mitose/fisiologia , Fosforilação , Processamento de Proteína Pós-Traducional/fisiologia
7.
Biochem Pharmacol ; 167: 33-43, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30910692

RESUMO

Mitosis ensures accurate segregation of duplicated DNA through tight regulation of chromosome condensation, bipolar spindle assembly, chromosome alignment in the metaphase plate, chromosome segregation and cytokinesis. Poly(ADP-ribose) polymerases (PARPs), in particular PARP1, PARP2, PARP3, PARP5a (TNKS1), as well as poly(ADP-ribose) glycohydrolase (PARG), regulate different mitotic functions, including centrosome function, mitotic spindle assembly, mitotic checkpoints, telomere length and telomere cohesion. PARP depletion or inhibition give rise to various mitotic defects such as centrosome amplification, multipolar spindles, chromosome misalignment, premature loss of cohesion, metaphase arrest, anaphase DNA bridges, lagging chromosomes, and micronuclei. As the mechanisms of PARP1/2 inhibitor-mediated cell death are being progressively elucidated, it is becoming clear that mitotic defects caused by PARP1/2 inhibition arise due to replication stress and DNA damage in S phase. As it stands, entrapment of inactive PARP1/2 on DNA phenocopies replication stress through accumulation of unresolved replication intermediates, double-stranded DNA breaks (DSBs) and incorrectly repaired DSBs, which can be transmitted from S phase to mitosis and instigate various mitotic defects, giving rise to both numerical and structural chromosomal aberrations. Cancer cells have increased levels of replication stress, which makes them particularly susceptible to a combination of agents that compromise replication fork stability. Indeed, combining PARP1/2 inhibitors with genetic deficiencies in DNA repair pathways, DNA-damaging agents, ATR and other cell cycle checkpoint inhibitors has yielded synergistic effects in killing cancer cells. Here I provide a comprehensive overview of the mitotic functions of PARPs and PARG, mitotic phenotypes induced by their depletion or inhibition, as well as the therapeutic relevance of targeting mitotic cells by directly interfering with mitotic functions or indirectly through replication stress.


Assuntos
Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Mitose/fisiologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Humanos , Mitose/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1/metabolismo
8.
Nucleus ; 9(1): 474-491, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30205747

RESUMO

Fluorescence microscopy in combination with the induction of localized DNA damage using focused light beams has played a major role in the study of protein recruitment kinetics to DNA damage sites in recent years. Currently published methods are dedicated to the study of single fluorophore/single protein kinetics. However, these methods may be limited when studying the relative recruitment dynamics between two or more proteins due to cell-to-cell variability in gene expression and recruitment kinetics, and are not suitable for comparative analysis of fast-recruiting proteins. To tackle these limitations, we have established a time-lapse fluorescence microscopy method based on simultaneous dual-channel acquisition following UV-A-induced local DNA damage coupled with a standardized image and recruitment analysis workflow. Simultaneous acquisition is achieved by spectrally splitting the emitted light into two light paths, which are simultaneously imaged on two halves of the same camera chip. To validate this method, we studied the recruitment of poly(ADP-ribose) polymerase 1 (PARP1), poly (ADP-ribose) glycohydrolase (PARG), proliferating cell nuclear antigen (PCNA) and the chromatin remodeler ALC1. In accordance with the published data based on single fluorophore imaging, simultaneous dual-channel imaging revealed that PARP1 regulates fast recruitment and dissociation of PARG and that in PARP1-depleted cells PARG and PCNA are recruited with comparable kinetics. This approach is particularly advantageous for analyzing the recruitment sequence of fast-recruiting proteins such as PARP1 and ALC1, and revealed that PARP1 is recruited faster than ALC1. Split-view imaging can be incorporated into any laser microirradiation-adapted microscopy setup together with a recruitment-dedicated image analysis package.


Assuntos
Dano ao DNA , DNA Helicases/análise , Proteínas de Ligação a DNA/análise , Glicosídeo Hidrolases/análise , Lasers , Imagem Óptica , Poli(ADP-Ribose) Polimerase-1/análise , Antígeno Nuclear de Célula em Proliferação/análise , Raios Ultravioleta , Linhagem Celular Tumoral , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Glicosídeo Hidrolases/metabolismo , Humanos , Cinética , Microscopia de Fluorescência , Poli(ADP-Ribose) Polimerase-1/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo
9.
Genes (Basel) ; 9(8)2018 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-30126151

RESUMO

DNA replication and repair are essential cellular processes that ensure genome duplication and safeguard the genome from deleterious mutations. Both processes utilize an abundance of enzymatic functions that need to be tightly regulated to ensure dynamic exchange of DNA replication and repair factors. Proliferating cell nuclear antigen (PCNA) is the major coordinator of faithful and processive replication and DNA repair at replication forks. Post-translational modifications of PCNA, ubiquitination and acetylation in particular, regulate the dynamics of PCNA-protein interactions. Proliferating cell nuclear antigen (PCNA) monoubiquitination elicits 'polymerase switching', whereby stalled replicative polymerase is replaced with a specialized polymerase, while PCNA acetylation may reduce the processivity of replicative polymerases to promote homologous recombination-dependent repair. While regulatory functions of PCNA ubiquitination and acetylation have been well established, the regulation of PCNA-binding proteins remains underexplored. Considering the vast number of PCNA-binding proteins, many of which have similar PCNA binding affinities, the question arises as to the regulation of the strength and sequence of their binding to PCNA. Here I provide an overview of post-translational modifications on both PCNA and PCNA-interacting proteins and discuss their relevance for the regulation of the dynamic processes of DNA replication and repair.

10.
Oncotarget ; 8(61): 103931-103951, 2017 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-29262611

RESUMO

Poly(ADP-ribose) polymerases (PARPs) regulate various aspects of cellular function including mitotic progression. Although PARP inhibitors have been undergoing various clinical trials and the PARP1/2 inhibitor olaparib was approved as monotherapy for BRCA-mutated ovarian cancer, their mode of action in killing tumour cells is not fully understood. We investigated the effect of PARP inhibition on mitosis in cancerous (cervical, ovary, breast and osteosarcoma) and non-cancerous cells by live-cell imaging. The clinically relevant inhibitor olaparib induced strong perturbations in mitosis, including problems with chromosome alignment at the metaphase plate, anaphase delay, and premature loss of cohesion (cohesion fatigue) after a prolonged metaphase arrest, resulting in sister chromatid scattering. PARP1 and PARP2 depletion suppressed the phenotype while PARP2 overexpression enhanced it, suggesting that olaparib-bound PARP1 and PARP2 rather than the lack of catalytic activity causes this phenotype. Olaparib-induced mitotic chromatid scattering was observed in various cancer cell lines with increased protein levels of PARP1 and PARP2, but not in non-cancer or cancer cell lines that expressed lower levels of PARP1 or PARP2. Interestingly, the sister chromatid scattering phenotype occurred only when olaparib was added during the S-phase preceding mitosis, suggesting that PARP1 and PARP2 entrapment at replication forks impairs sister chromatid cohesion. Clinically relevant DNA-damaging agents that impair replication progression such as topoisomerase inhibitors and cisplatin were also found to induce sister chromatid scattering and metaphase plate alignment problems, suggesting that these mitotic phenotypes are a common outcome of replication perturbation.

11.
Nucleic Acids Res ; 45(16): 9741-9759, 2017 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-28934471

RESUMO

Poly(ADP-ribose) glycohydrolase (PARG) regulates cellular poly(ADP-ribose) (PAR) levels by rapidly cleaving glycosidic bonds between ADP-ribose units. PARG interacts with proliferating cell nuclear antigen (PCNA) and is strongly recruited to DNA damage sites in a PAR- and PCNA-dependent fashion. Here we identified PARG acetylation site K409 that is essential for its interaction with PCNA, its localization within replication foci and its recruitment to DNA damage sites. We found K409 to be part of a non-canonical PIP-box within the PARG disordered regulatory region. The previously identified putative N-terminal PIP-box does not bind PCNA directly but contributes to PARG localization within replication foci. X-ray structure and MD simulations reveal that the PARG non-canonical PIP-box binds PCNA in a manner similar to other canonical PIP-boxes and may represent a new type of PIP-box. While the binding of previously described PIP-boxes is based on hydrophobic interactions, PARG PIP-box binds PCNA via both stabilizing hydrophobic and fine-tuning electrostatic interactions. Our data explain the mechanism of PARG-PCNA interaction through a new PARG PIP-box that exhibits non-canonical sequence properties but a canonical mode of PCNA binding.


Assuntos
Glicosídeo Hidrolases/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Acetilação , Calorimetria/métodos , Cromatina/metabolismo , Cristalografia por Raios X , Dano ao DNA , Transferência Ressonante de Energia de Fluorescência , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Células HeLa , Humanos , Imunoprecipitação , Lasers , Lisina/genética , Lisina/metabolismo , Simulação de Dinâmica Molecular , Antígeno Nuclear de Célula em Proliferação/química , Conformação Proteica , Fase S/genética , Eletricidade Estática
12.
J Biol Chem ; 292(43): 17643-17657, 2017 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-28864776

RESUMO

N-terminal histone tails are subject to many posttranslational modifications that are recognized by and interact with designated reader domains in histone-binding proteins. BROMO domain adjacent to zinc finger 2B (BAZ2B) is a multidomain histone-binding protein that contains two histone reader modules, a plant homeodomain (PHD) and a bromodomain (BRD), linked by a largely disordered linker. Although previous studies have reported specificity of the PHD domain for the unmodified N terminus of histone H3 and of the BRD domain for H3 acetylated at Lys14 (H3K14ac), the exact mode of H3 binding by BAZ2B and its regulation are underexplored. Here, using isothermal titration calorimetry and NMR spectroscopy, we report that acidic residues in the BAZ2B PHD domain are essential for H3 binding and that BAZ2B PHD-BRD establishes a polyvalent interaction with H3K14ac. Furthermore, we provide evidence that the disordered interdomain linker modulates the histone-binding affinity by interacting with the PHD domain. In particular, lysine-rich stretches in the linker, which resemble the positively charged N terminus of histone H3, reduce the binding affinity of the PHD finger toward the histone substrate. Phosphorylation, acetylation, or poly(ADP-ribosyl)ation of the linker residues may therefore act as a cellular mechanism to transiently tune BAZ2B histone-binding affinity. Our findings further support the concept of interdomain linkers serving a dual role in substrate binding by appropriately positioning the adjacent domains and by electrostatically modulating substrate binding. Moreover, inhibition of histone binding by a histone-mimicking interdomain linker represents another example of regulation of protein-protein interactions by intramolecular mimicry.


Assuntos
Histonas/química , Proteínas/química , Histonas/genética , Histonas/metabolismo , Humanos , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas/genética , Proteínas/metabolismo , Fatores Genéricos de Transcrição
13.
J Cell Sci ; 129(24): 4607-4621, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27875273

RESUMO

Sirtuin 2 (SIRT2) is an NAD-dependent deacetylase known to regulate microtubule dynamics and cell cycle progression. SIRT2 has also been implicated in the pathology of cancer, neurodegenerative diseases and progeria. Here, we show that SIRT2 depletion or overexpression causes nuclear envelope reassembly defects. We link this phenotype to the recently identified regulator of nuclear envelope reassembly ANKLE2. ANKLE2 acetylation at K302 and phosphorylation at S662 are dynamically regulated throughout the cell cycle by SIRT2 and are essential for normal nuclear envelope reassembly. The function of SIRT2 therefore extends beyond the regulation of microtubules to include the regulation of nuclear envelope dynamics.


Assuntos
Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Sirtuína 2/metabolismo , Acetilação , Biotinilação , Ciclo Celular , Forma do Núcleo Celular , Cromatografia de Afinidade , Células HEK293 , Humanos , Modelos Biológicos , Fosforilação , Ligação Proteica , Proteômica
14.
Nature ; 477(7366): 616-20, 2011 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-21892188

RESUMO

Post-translational modification of proteins by poly(ADP-ribosyl)ation regulates many cellular pathways that are critical for genome stability, including DNA repair, chromatin structure, mitosis and apoptosis. Poly(ADP-ribose) (PAR) is composed of repeating ADP-ribose units linked via a unique glycosidic ribose-ribose bond, and is synthesized from NAD by PAR polymerases. PAR glycohydrolase (PARG) is the only protein capable of specific hydrolysis of the ribose-ribose bonds present in PAR chains; its deficiency leads to cell death. Here we show that filamentous fungi and a number of bacteria possess a divergent form of PARG that has all the main characteristics of the human PARG enzyme. We present the first PARG crystal structure (derived from the bacterium Thermomonospora curvata), which reveals that the PARG catalytic domain is a distant member of the ubiquitous ADP-ribose-binding macrodomain family. High-resolution structures of T. curvata PARG in complexes with ADP-ribose and the PARG inhibitor ADP-HPD, complemented by biochemical studies, allow us to propose a model for PAR binding and catalysis by PARG. The insights into the PARG structure and catalytic mechanism should greatly improve our understanding of how PARG activity controls reversible protein poly(ADP-ribosyl)ation and potentially of how the defects in this regulation are linked to human disease.


Assuntos
Actinomycetales/enzimologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Adenosina Difosfato Ribose/química , Adenosina Difosfato Ribose/metabolismo , Sequência de Aminoácidos , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Evolução Molecular , Glicosídeo Hidrolases/antagonistas & inibidores , Glicosídeo Hidrolases/genética , Humanos , Hidrólise , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Poli(ADP-Ribose) Polimerase-1 , Poli Adenosina Difosfato Ribose/química , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Conformação Proteica , Proteínas/metabolismo , Pirrolidinas/farmacologia
15.
Microbiol Mol Biol Rev ; 75(1): 133-91, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21372322

RESUMO

Deinococcus radiodurans is a robust bacterium best known for its capacity to repair massive DNA damage efficiently and accurately. It is extremely resistant to many DNA-damaging agents, including ionizing radiation and UV radiation (100 to 295 nm), desiccation, and mitomycin C, which induce oxidative damage not only to DNA but also to all cellular macromolecules via the production of reactive oxygen species. The extreme resilience of D. radiodurans to oxidative stress is imparted synergistically by an efficient protection of proteins against oxidative stress and an efficient DNA repair mechanism, enhanced by functional redundancies in both systems. D. radiodurans assets for the prevention of and recovery from oxidative stress are extensively reviewed here. Radiation- and desiccation-resistant bacteria such as D. radiodurans have substantially lower protein oxidation levels than do sensitive bacteria but have similar yields of DNA double-strand breaks. These findings challenge the concept of DNA as the primary target of radiation toxicity while advancing protein damage, and the protection of proteins against oxidative damage, as a new paradigm of radiation toxicity and survival. The protection of DNA repair and other proteins against oxidative damage is imparted by enzymatic and nonenzymatic antioxidant defense systems dominated by divalent manganese complexes. Given that oxidative stress caused by the accumulation of reactive oxygen species is associated with aging and cancer, a comprehensive outlook on D. radiodurans strategies of combating oxidative stress may open new avenues for antiaging and anticancer treatments. The study of the antioxidation protection in D. radiodurans is therefore of considerable potential interest for medicine and public health.


Assuntos
Deinococcus/fisiologia , Estresse Oxidativo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dano ao DNA , Reparo do DNA , Deinococcus/genética , Deinococcus/patogenicidade , Deinococcus/efeitos da radiação , Estresse Oxidativo/fisiologia , Tolerância a Radiação , Espécies Reativas de Oxigênio/metabolismo
16.
J Biol Chem ; 286(15): 13261-71, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21257746

RESUMO

Sirtuins are a family of protein lysine deacetylases, which regulate gene silencing, metabolism, life span, and chromatin structure. Sirtuins utilize NAD(+) to deacetylate proteins, yielding O-acetyl-ADP-ribose (OAADPr) as a reaction product. The macrodomain is a ubiquitous protein module known to bind ADP-ribose derivatives, which diverged through evolution to support many different protein functions and pathways. The observation that some sirtuins and macrodomains are physically linked as fusion proteins or genetically coupled through the same operon, provided a clue that their functions might be connected. Indeed, here we demonstrate that the product of the sirtuin reaction OAADPr is a substrate for several related macrodomain proteins: human MacroD1, human MacroD2, Escherichia coli YmdB, and the sirtuin-linked MacroD-like protein from Staphylococcus aureus. In addition, we show that the cell extracts derived from MacroD-deficient Neurospora crassa strain exhibit a major reduction in the ability to hydrolyze OAADPr. Our data support a novel function of macrodomains as OAADPr deacetylases and potential in vivo regulators of cellular OAADPr produced by NAD(+)-dependent deacetylation.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Evolução Molecular , Proteínas Fúngicas/química , Neurospora crassa/enzimologia , Sirtuínas/química , Staphylococcus aureus/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Células HeLa , Humanos , Neurospora crassa/genética , Estrutura Terciária de Proteína , Sirtuínas/genética , Sirtuínas/metabolismo , Staphylococcus aureus/genética
17.
DNA Repair (Amst) ; 9(11): 1151-61, 2010 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-20817622

RESUMO

Deinococcus radiodurans is one of the most radiation-resistant organisms known. It can repair hundreds of radiation-induced double-strand DNA breaks without loss of viability. Genome reassembly in heavily irradiated D. radiodurans is considered to be an error-free process since no genome rearrangements were detected after post-irradiation repair. Here, we describe for the first time conditions that frequently cause erroneous chromosomal assemblies. Gross chromosomal rearrangements have been detected in recA mutant cells that survived exposure to 5kGy γ-radiation. The recA mutants are prone also to spontaneous DNA rearrangements during normal exponential growth. Some insertion sequences have been identified as dispersed genomic homology blocks that can mediate DNA rearrangements. Whereas the wild-type D. radiodurans appears to repair accurately its genome shattered by 5kGy γ-radiation, extremely high γ-doses, e.g., 25kGy, produce frequent genome rearrangements among survivors. Our results show that the RecA protein is quintessential for the fidelity of repair of both spontaneous and γ-radiation-induced DNA breaks and, consequently, for genome stability in D. radiodurans. The mechanisms of decreased genome stability in the absence of RecA are discussed.


Assuntos
Reparo do DNA , Deinococcus/enzimologia , Deinococcus/genética , Instabilidade Genômica , Recombinases Rec A/metabolismo , Proliferação de Células/efeitos da radiação , Quebras de DNA/efeitos da radiação , Fragmentação do DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Deinococcus/citologia , Deinococcus/efeitos da radiação , Relação Dose-Resposta à Radiação , Raios gama , Rearranjo Gênico/efeitos da radiação , Genoma Bacteriano/genética , Instabilidade Genômica/efeitos da radiação , Mutação/efeitos da radiação , Recombinases Rec A/genética
18.
Cell ; 136(6): 1044-55, 2009 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-19303848

RESUMO

Deinococcus radiodurans' extreme resistance to ionizing radiation, desiccation, and DNA-damaging chemicals involves a robust DNA repair that reassembles its shattered genome. The repair process requires diploidy and commences with an extensive exonucleolytic erosion of DNA fragments. Liberated single-stranded overhangs prime strand elongation on overlapping fragments and the elongated complementary strands reestablish chromosomal contiguity by annealing. We explored the interdependence of the DNA recombination and replication processes in the reconstitution of the D. radiodurans genome disintegrated by ionizing radiation. The priming of extensive DNA repair synthesis involves RecA and RadA proteins. DNA polymerase III is essential for the initiation of repair synthesis, whereas efficient elongation requires DNA polymerases I and III. Inactivation of both polymerases leads to degradation of DNA fragments and rapid cell death. The present in vivo characterization of key recombination and replication processes dissects the mechanism of DNA repair in heavily irradiated D. radiodurans.


Assuntos
Reparo do DNA , Deinococcus/genética , Deinococcus/efeitos da radiação , Recombinação Genética , Proteínas de Bactérias/metabolismo , Dano ao DNA , DNA Polimerase III , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA , Deinococcus/enzimologia , Deinococcus/metabolismo , Raios gama , Genoma Bacteriano , Hidroxiureia/farmacologia , Recombinases Rec A/metabolismo
19.
Genetics ; 176(3): 1431-40, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17483416

RESUMO

Escherichia coli PolIV, a DNA polymerase capable of catalyzing synthesis past replication-blocking DNA lesions, belongs to the most ubiquitous branch of Y-family DNA polymerases. The goal of this study is to identify spontaneous DNA damage that is bypassed specifically and accurately by PolIV in vivo. We increased the amount of spontaneous DNA lesions using mutants deficient for different DNA repair pathways and measured mutation frequency in PolIV-proficient and -deficient backgrounds. We found that PolIV performs an error-free bypass of DNA damage that accumulates in the alkA tag genetic background. This result indicates that PolIV is involved in the error-free bypass of cytotoxic alkylating DNA lesions. When the amount of cytotoxic alkylating DNA lesions is increased by the treatment with chemical alkylating agents, PolIV is required for survival in an alkA tag-proficient genetic background as well. Our study, together with the reported involvement of the mammalian PolIV homolog, Polkappa, in similar activity, indicates that Y-family DNA polymerases from the DinB branch can be added to the list of evolutionarily conserved molecular mechanisms that counteract cytotoxic effects of DNA alkylation. This activity is of major biological relevance because alkylating agents are continuously produced endogenously in all living cells and are also present in the environment.


Assuntos
Adutos de DNA , Dano ao DNA , DNA Polimerase beta/fisiologia , Alquilantes/farmacologia , Escherichia coli/genética , Mutação
20.
Nature ; 443(7111): 569-73, 2006 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-17006450

RESUMO

Dehydration or desiccation is one of the most frequent and severe challenges to living cells. The bacterium Deinococcus radiodurans is the best known extremophile among the few organisms that can survive extremely high exposures to desiccation and ionizing radiation, which shatter its genome into hundreds of short DNA fragments. Remarkably, these fragments are readily reassembled into a functional 3.28-megabase genome. Here we describe the relevant two-stage DNA repair process, which involves a previously unknown molecular mechanism for fragment reassembly called 'extended synthesis-dependent strand annealing' (ESDSA), followed and completed by crossovers. At least two genome copies and random DNA breakage are requirements for effective ESDSA. In ESDSA, chromosomal fragments with overlapping homologies are used both as primers and as templates for massive synthesis of complementary single strands, as occurs in a single-round multiplex polymerase chain reaction. This synthesis depends on DNA polymerase I and incorporates more nucleotides than does normal replication in intact cells. Newly synthesized complementary single-stranded extensions become 'sticky ends' that anneal with high precision, joining together contiguous DNA fragments into long, linear, double-stranded intermediates. These intermediates require RecA-dependent crossovers to mature into circular chromosomes that comprise double-stranded patchworks of numerous DNA blocks synthesized before radiation, connected by DNA blocks synthesized after radiation.


Assuntos
Cromossomos Bacterianos/genética , Dano ao DNA , Reparo do DNA/genética , Deinococcus/genética , Bromodesoxiuridina/metabolismo , Cromossomos Bacterianos/efeitos da radiação , Dano ao DNA/efeitos da radiação , DNA Polimerase I/metabolismo , Reparo do DNA/efeitos da radiação , Replicação do DNA , DNA Bacteriano/biossíntese , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Bacteriano/efeitos da radiação , Deinococcus/efeitos da radiação , Dessecação , Genoma Bacteriano , Modelos Genéticos , Fotólise/efeitos da radiação , Tolerância a Radiação
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