Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 884
Filtrar
1.
Pediatrics ; 146(Suppl 1): S60-S65, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32737234

RESUMO

Charlie Gard (August 4, 2016, to July 28, 2017) was an infant in the United Kingdom who was diagnosed with an encephalopathic form of mitochondrial DNA depletion syndrome caused by a mutation in the RRM2B gene. Charlie's parents raised £1.3 million (∼$1.6 million US) on a crowdfunding platform to travel to New York to pursue experimental nucleoside bypass treatment, which was being used to treat a myopathic form of mitochondrial DNA depletion syndrome caused by mutations in a different gene (TK2). The case made international headlines about what was in Charlie's best interest. In the medical ethics community, it raised the question of whether best interest serves as a guidance principle (a principle that provides substantive directions as to how decisions are to be made), an intervention principle (a principle specifying the conditions under which third parties are to intervene), both guidance and intervention, or neither. I show that the United Kingdom uses best interest as both guidance and intervention, and the United States uses best interest for neither. This explains why the decision to withdraw the ventilator without attempting nucleoside bypass treatment was the correct decision in the United Kingdom and why the opposite conclusion would have been reached in the United States.


Assuntos
Proteínas de Ciclo Celular/genética , Encefalomiopatias Mitocondriais/terapia , Defesa do Paciente/ética , Respiração Artificial/ética , Ribonucleotídeo Redutases/genética , Suspensão de Tratamento/ética , Tomada de Decisão Clínica/ética , Crowdsourcing/economia , História do Século XXI , Humanos , Lactente , Masculino , Futilidade Médica/ética , Encefalomiopatias Mitocondriais/genética , Cidade de Nova Iorque , Poder Familiar , Defesa do Paciente/legislação & jurisprudência , Transferência de Pacientes/ética , Transferência de Pacientes/legislação & jurisprudência , Guias de Prática Clínica como Assunto , Timidina Quinase/genética , Reino Unido , Estados Unidos , Suspensão de Tratamento/legislação & jurisprudência
2.
Science ; 368(6489): 424-427, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32217749

RESUMO

Ribonucleotide reductases (RNRs) are a diverse family of enzymes that are alone capable of generating 2'-deoxynucleotides de novo and are thus critical in DNA biosynthesis and repair. The nucleotide reduction reaction in all RNRs requires the generation of a transient active site thiyl radical, and in class I RNRs, this process involves a long-range radical transfer between two subunits, α and ß. Because of the transient subunit association, an atomic resolution structure of an active α2ß2 RNR complex has been elusive. We used a doubly substituted ß2, E52Q/(2,3,5)-trifluorotyrosine122-ß2, to trap wild-type α2 in a long-lived α2ß2 complex. We report the structure of this complex by means of cryo-electron microscopy to 3.6-angstrom resolution, allowing for structural visualization of a 32-angstrom-long radical transfer pathway that affords RNR activity.


Assuntos
Proteínas de Escherichia coli/química , Ribonucleotídeo Redutases/química , Biocatálise , Domínio Catalítico , Microscopia Crioeletrônica , Proteínas de Escherichia coli/genética , Holoenzimas/química , Holoenzimas/genética , Conformação Proteica , Ribonucleotídeo Redutases/genética , Tirosina/química
3.
Nucleic Acids Res ; 48(8): 4274-4297, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32187369

RESUMO

Cellular levels of ribonucleoside triphosphates (rNTPs) are much higher than those of deoxyribonucleoside triphosphates (dNTPs), thereby influencing the frequency of incorporation of ribonucleoside monophosphates (rNMPs) by DNA polymerases (Pol) into DNA. RNase H2-initiated ribonucleotide excision repair (RER) efficiently removes single rNMPs in genomic DNA. However, processing of rNMPs by Topoisomerase 1 (Top1) in absence of RER induces mutations and genome instability. Here, we greatly increased the abundance of genomic rNMPs in Saccharomyces cerevisiae by depleting Rnr1, the major subunit of ribonucleotide reductase, which converts ribonucleotides to deoxyribonucleotides. We found that in strains that are depleted of Rnr1, RER-deficient, and harbor an rNTP-permissive replicative Pol mutant, excessive accumulation of single genomic rNMPs severely compromised growth, but this was reversed in absence of Top1. Thus, under Rnr1 depletion, limited dNTP pools slow DNA synthesis by replicative Pols and provoke the incorporation of high levels of rNMPs in genomic DNA. If a threshold of single genomic rNMPs is exceeded in absence of RER and presence of limited dNTP pools, Top1-mediated genome instability leads to severe growth defects. Finally, we provide evidence showing that accumulation of RNA/DNA hybrids in absence of RNase H1 and RNase H2 leads to cell lethality under Rnr1 depletion.


Assuntos
DNA Topoisomerases Tipo I/metabolismo , Ribonucleotídeo Redutases/genética , Ribonucleotídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Dano ao DNA , Desoxirribonucleotídeos/metabolismo , Genoma Fúngico , Instabilidade Genômica , Mutação , Ribonuclease H/genética , Ribonucleases/genética , Pontos de Checagem da Fase S do Ciclo Celular , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Deleção de Sequência
4.
Nat Microbiol ; 5(2): 331-342, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31844296

RESUMO

Viruses manipulate cellular signalling by inducing the degradation of crucial signal transducers, usually via the ubiquitin-proteasome pathway. Here, we show that the murine cytomegalovirus (Murid herpesvirus 1) M45 protein induces the degradation of two cellular signalling proteins, the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) essential modulator (NEMO) and the receptor-interacting protein kinase 1 (RIPK1), via a different mechanism: it induces their sequestration as insoluble protein aggregates and subsequently facilitates their degradation by autophagy. Aggregation of target proteins requires a distinct sequence motif in M45, which we termed 'induced protein aggregation motif'. In a second step, M45 recruits the retromer component vacuolar protein sorting 26B (VPS26B) and the microtubule-associated protein light chain 3 (LC3)-interacting adaptor protein TBC1D5 to facilitate degradation of aggregates by selective autophagy. The induced protein aggregation motif is conserved in M45-homologous proteins of several human herpesviruses, including herpes simplex virus, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, but is only partially conserved in the human cytomegalovirus UL45 protein. We further show that the HSV-1 ICP6 protein induces RIPK1 aggregation and degradation in a similar fashion to M45. These data suggest that induced protein aggregation combined with selective autophagy of aggregates (aggrephagy) represents a conserved viral immune-evasion mechanism.


Assuntos
Herpesviridae/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologia , Animais , Autofagia/imunologia , Proteína 5 Relacionada à Autofagia/deficiência , Proteína 5 Relacionada à Autofagia/genética , Células Cultivadas , Células HEK293 , Herpesviridae/metabolismo , Herpesviridae/patogenicidade , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 1/patogenicidade , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Evasão da Resposta Imune , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Muromegalovirus/imunologia , Muromegalovirus/metabolismo , Muromegalovirus/patogenicidade , Agregados Proteicos/imunologia , Proteólise , Proteína Serina-Treonina Quinases de Interação com Receptores/química , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/imunologia , Ribonucleotídeo Redutases/metabolismo , Proteínas Virais/genética , Proteínas Virais/imunologia , Proteínas Virais/metabolismo
5.
Microb Cell Fact ; 18(1): 179, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31640713

RESUMO

BACKGROUND: Pseudomonas putida is a metabolically versatile, genetically accessible, and stress-robust species with outstanding potential to be used as a workhorse for industrial applications. While industry recognises the importance of robustness under micro-oxic conditions for a stable production process, the obligate aerobic nature of P. putida, attributed to its inability to produce sufficient ATP and maintain its redox balance without molecular oxygen, severely limits its use for biotechnology applications. RESULTS: Here, a combination of genome-scale metabolic modelling and comparative genomics is used to pinpoint essential [Formula: see text]-dependent processes. These explain the inability of the strain to grow under anoxic conditions: a deficient ATP generation and an inability to synthesize essential metabolites. Based on this, several P. putida recombinant strains were constructed harbouring acetate kinase from Escherichia coli for ATP production, and a class I dihydroorotate dehydrogenase and a class III anaerobic ribonucleotide triphosphate reductase from Lactobacillus lactis for the synthesis of essential metabolites. Initial computational designs were fine-tuned by means of adaptive laboratory evolution. CONCLUSIONS: We demonstrated the value of combining in silico approaches, experimental validation and adaptive laboratory evolution for microbial design by making the strictly aerobic Pseudomonas putida able to grow under micro-oxic conditions.


Assuntos
Proteínas de Bactérias/genética , Microrganismos Geneticamente Modificados , Oxigênio/metabolismo , Pseudomonas putida , Acetato Quinase/genética , Acetato Quinase/metabolismo , Anaerobiose , Proteínas de Bactérias/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Genômica , Lactobacillus/enzimologia , Lactobacillus/metabolismo , Engenharia Metabólica , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismo
6.
PLoS Genet ; 15(8): e1008136, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31381575

RESUMO

The S-phase checkpoint plays an essential role in regulation of the ribonucleotide reductase (RNR) activity to maintain the dNTP pools. How eukaryotic cells respond appropriately to different levels of replication threats remains elusive. Here, we have identified that a conserved GSK-3 kinase Mck1 cooperates with Dun1 in regulating this process. Deleting MCK1 sensitizes dun1Δ to hydroxyurea (HU) reminiscent of mec1Δ or rad53Δ. While Mck1 is downstream of Rad53, it does not participate in the post-translational regulation of RNR as Dun1 does. Mck1 phosphorylates and releases the Crt1 repressor from the promoters of DNA damage-inducible genes as RNR2-4 and HUG1. Hug1, an Rnr2 inhibitor normally silenced, is induced as a counterweight to excessive RNR. When cells suffer a more severe threat, Mck1 inhibits HUG1 transcription. Consistently, only a combined deletion of HUG1 and CRT1, confers a dramatic boost of dNTP levels and the survival of mck1Δdun1Δ or mec1Δ cells assaulted by a lethal dose of HU. These findings reveal the division-of-labor between Mck1 and Dun1 at the S-phase checkpoint pathway to fine-tune dNTP homeostasis.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Regulação Fúngica da Expressão Gênica/fisiologia , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Ciclo Celular/genética , Dano ao DNA , Replicação do DNA/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Quinase 3 da Glicogênio Sintase/genética , Hidroxiureia/toxicidade , Nucleotídeos/metabolismo , Fosforilação , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética
7.
Nat Commun ; 10(1): 3213, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324785

RESUMO

Ribonucleotide reductase (RNR) catalyzes the de novo synthesis of deoxyribonucleoside diphosphates (dNDPs) to provide dNTP precursors for DNA synthesis. Here, we report that acetylation and deacetylation of the RRM2 subunit of RNR acts as a molecular switch that impacts RNR activity, dNTP synthesis, and DNA replication fork progression. Acetylation of RRM2 at K95 abrogates RNR activity by disrupting its homodimer assembly. RRM2 is directly acetylated by KAT7, and deacetylated by Sirt2, respectively. Sirt2, which level peak in S phase, sustains RNR activity at or above a threshold level required for dNTPs synthesis. We also find that radiation or camptothecin-induced DNA damage promotes RRM2 deacetylation by enhancing Sirt2-RRM2 interaction. Acetylation of RRM2 at K95 results in the reduction of the dNTP pool, DNA replication fork stalling, and the suppression of tumor cell growth in vitro and in vivo. This study therefore identifies acetylation as a regulatory mechanism governing RNR activity.


Assuntos
Transformação Celular Neoplásica/metabolismo , Ribonucleotídeo Redutases/metabolismo , Acetilação , Camptotecina/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Dano ao DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Histona Acetiltransferases/metabolismo , Humanos , Ribonucleosídeo Difosfato Redutase/metabolismo , Ribonucleotídeo Redutases/genética , Fase S/efeitos dos fármacos , Sirtuína 2/metabolismo
8.
Nat Commun ; 10(1): 2653, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201319

RESUMO

Ribonucleotide reductases (RNRs) use a conserved radical-based mechanism to catalyze the conversion of ribonucleotides to deoxyribonucleotides. Within the RNR family, class Ib RNRs are notable for being largely restricted to bacteria, including many pathogens, and for lacking an evolutionarily mobile ATP-cone domain that allosterically controls overall activity. In this study, we report the emergence of a distinct and unexpected mechanism of activity regulation in the sole RNR of the model organism Bacillus subtilis. Using a hypothesis-driven structural approach that combines the strengths of small-angle X-ray scattering (SAXS), crystallography, and cryo-electron microscopy (cryo-EM), we describe the reversible interconversion of six unique structures, including a flexible active tetramer and two inhibited helical filaments. These structures reveal the conformational gymnastics necessary for RNR activity and the molecular basis for its control via an evolutionarily convergent form of allostery.


Assuntos
Sítio Alostérico/genética , Proteínas de Bactérias/genética , Ribonucleotídeo Redutases/genética , Regulação Alostérica/genética , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , Cristalografia por Raios X , Evolução Molecular , Modelos Moleculares , Estrutura Quaternária de Proteína/genética , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Ribonucleotídeo Redutases/ultraestrutura , Ribonucleotídeos/metabolismo , Espalhamento a Baixo Ângulo
9.
Int J Pediatr Otorhinolaryngol ; 121: 143-149, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30909120

RESUMO

OBJECTIVES: Although hearing loss is a well-known symptom of mitochondria-related disorders, it is not clear how often it is a congenital and cochlear impairment. The Newborn Hearing Screening Program (NHSP) enables to distinguish congenital cochlear deafness from an acquired hearing deficit. The initial aim of the study was to research the frequency of the congenital cochlear hearing loss among patients with various gene defects resulting in mitochondrial disorders. The research process brought on an additional gain: basing on our preliminary study group of 80 patients, in 12 patients altogether we identified two defected genes responsible for mitochondrial disorders, whose carriers did not pass the NHSP. Finally, these patients were diagnosed with the congenital cochlear deafness. MATERIAL AND METHODS: The results of the NHSP in the patients with mitochondrial disorders diagnosed in our tertiary reference center were analyzed. Only the cases with confirmed mutations were qualified for the study group. The NHSP database included 80 patients with mutations in 31 different genes: 25 nuclear-encoded and 6 mtDNA-encoded. We searched the literature for the presence of a congenital hearing impairment (CHI) in mitochondrial disorders caused by changes in 278 already known genes. RESULTS: For 68 patients from the study group the NHSP test indicated a proper cochlear function and thus suggested normal hearing. For 12 mitochondrial patients, the NHSP test indicated the requirement for the further audiological diagnosis, and finally CHI was confirmed in 8 of them. This latter subset included patients with pathogenic variants in RRM2B and SERAC1, known as "deafness-causing genes". Contrary to our initial expectations, the patients carrying mutations in other "deafness-causing genes": MPV17, POLG, COX10, as well as other mitochondria-related genes, all reported in literature, did not indicate any CHI following the NHSP test. CONCLUSION: Our study indicates that the cochlear CHI is a phenotypic feature of the RRM2B and SERAC1 related defects. The diagnosis of the CHI following the NHSP allows to early distinguish those defects from other mitochondria-related disorders in which the NHSP test result is correct. Wider studies are needed to assess the significance of this observation.


Assuntos
Hidrolases de Éster Carboxílico/genética , Proteínas de Ciclo Celular/genética , Surdez/genética , Perda Auditiva Neurossensorial/genética , Doenças Mitocondriais/genética , Ribonucleotídeo Redutases/genética , Adolescente , Criança , Pré-Escolar , DNA Mitocondrial , Surdez/congênito , Feminino , Perda Auditiva Neurossensorial/congênito , Testes Auditivos , Heterozigoto , Hospitais , Humanos , Lactente , Recém-Nascido , Masculino , Doenças Mitocondriais/complicações , Doenças Mitocondriais/diagnóstico , Mutação , Triagem Neonatal , Polônia
10.
Biochemistry ; 58(14): 1845-1860, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30855138

RESUMO

Class I ribonucleotide reductases (RNRs) share a common mechanism of nucleotide reduction in a catalytic α subunit. All RNRs initiate catalysis with a thiyl radical, generated in class I enzymes by a metallocofactor in a separate ß subunit. Class Id RNRs use a simple mechanism of cofactor activation involving oxidation of a MnII2 cluster by free superoxide to yield a metal-based MnIIIMnIV oxidant. This simple cofactor assembly pathway suggests that class Id RNRs may be representative of the evolutionary precursors to more complex class Ia-c enzymes. X-ray crystal structures of two class Id α proteins from Flavobacterium johnsoniae ( Fj) and Actinobacillus ureae ( Au) reveal that this subunit is distinctly small. The enzyme completely lacks common N-terminal ATP-cone allosteric motifs that regulate overall activity, a process that normally occurs by dATP-induced formation of inhibitory quaternary structures to prevent productive ß subunit association. Class Id RNR activity is insensitive to dATP in the Fj and Au enzymes evaluated here, as expected. However, the class Id α protein from Fj adopts higher-order structures, detected crystallographically and in solution. The Au enzyme does not exhibit these quaternary forms. Our study reveals structural similarity between bacterial class Id and eukaryotic class Ia α subunits in conservation of an internal auxiliary domain. Our findings with the Fj enzyme illustrate that nucleotide-independent higher-order quaternary structures can form in simple RNRs with truncated or missing allosteric motifs.


Assuntos
Domínio Catalítico , Desoxirribonucleotídeos/química , Conformação Proteica , Ribonucleotídeo Redutases/química , Actinobacillus/enzimologia , Actinobacillus/genética , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Biocatálise , Cristalografia por Raios X , Desoxirribonucleotídeos/biossíntese , Desoxirribonucleotídeos/genética , Flavobacterium/enzimologia , Flavobacterium/genética , Modelos Moleculares , Filogenia , Ribonucleotídeo Redutases/classificação , Ribonucleotídeo Redutases/genética , Espalhamento a Baixo Ângulo , Homologia de Sequência de Aminoácidos , Difração de Raios X
11.
J Biol Inorg Chem ; 24(2): 211-221, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30689052

RESUMO

R2-like ligand-binding oxidases (R2lox) assemble a heterodinuclear Mn/Fe cofactor which performs reductive dioxygen (O2) activation, catalyzes formation of a tyrosine-valine ether cross-link in the protein scaffold, and binds a fatty acid in a putative substrate channel. We have previously shown that the N-terminal metal binding site 1 is unspecific for manganese or iron in the absence of O2, but prefers manganese in the presence of O2, whereas the C-terminal site 2 is specific for iron. Here, we analyze the effects of amino acid exchanges in the cofactor environment on cofactor assembly and metalation specificity using X-ray crystallography, X-ray absorption spectroscopy, and metal quantification. We find that exchange of either the cross-linking tyrosine or the valine, regardless of whether the mutation still allows cross-link formation or not, results in unspecific manganese or iron binding at site 1 both in the absence or presence of O2, while site 2 still prefers iron as in the wild-type. In contrast, a mutation that blocks binding of the fatty acid does not affect the metal specificity of either site under anoxic or aerobic conditions, and cross-link formation is still observed. All variants assemble a dinuclear trivalent metal cofactor in the aerobic resting state, independently of cross-link formation. These findings imply that the cross-link residues are required to achieve the preference for manganese in site 1 in the presence of O2. The metalation specificity, therefore, appears to be established during the redox reactions leading to cross-link formation.


Assuntos
Reagentes para Ligações Cruzadas/metabolismo , Ferro/metabolismo , Manganês/metabolismo , Ribonucleotídeo Redutases/metabolismo , Tirosina/metabolismo , Valina/metabolismo , Reagentes para Ligações Cruzadas/química , Geobacillus/enzimologia , Ferro/química , Manganês/química , Mutação Puntual , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/genética , Tirosina/química , Valina/química
12.
Nucleic Acids Res ; 47(1): 237-252, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30462295

RESUMO

The balance and the overall concentration of intracellular deoxyribonucleoside triphosphates (dNTPs) are important determinants of faithful DNA replication. Despite the established fact that changes in dNTP pools negatively influence DNA replication fidelity, it is not clear why certain dNTP pool alterations are more mutagenic than others. As intracellular dNTP pools are mainly controlled by ribonucleotide reductase (RNR), and given the limited number of eukaryotic RNR mutations characterized so far, we screened for RNR1 mutations causing mutator phenotypes in Saccharomyces cerevisiae. We identified 24 rnr1 mutant alleles resulting in diverse mutator phenotypes linked in most cases to imbalanced dNTPs. Among the identified rnr1 alleles the strongest mutators presented a dNTP imbalance in which three out of the four dNTPs were elevated (dCTP, dTTP and dGTP), particularly if dGTP levels were highly increased. These rnr1 alleles caused growth defects/lethality in DNA replication fidelity-compromised backgrounds, and caused strong mutator phenotypes even in the presence of functional DNA polymerases and mismatch repair. In summary, this study pinpoints key residues that contribute to allosteric regulation of RNR's overall activity or substrate specificity. We propose a model that distinguishes between different dNTP pool alterations and provides a mechanistic explanation why certain dNTP imbalances are particularly detrimental.


Assuntos
Replicação do DNA/genética , Desoxirribonucleotídeos/genética , Ribonucleotídeo Redutases/genética , Proteínas de Saccharomyces cerevisiae/genética , Alelos , Reparo de Erro de Pareamento de DNA/genética , DNA Polimerase Dirigida por DNA/genética , Homeostase , Mutação/genética , Saccharomyces cerevisiae/genética
13.
FASEB J ; 33(2): 2017-2025, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30199284

RESUMO

Cellular supply of deoxythymidine triphosphate (dTTP) is crucial for DNA replication and repair. Thymidylate kinase (TMPK) catalyzes the conversion of thymidine monophosphate to thymidine diphosphate, which is an essential step for dTTP synthesis. Despite their major cellular localization in cytosol, TMPK and ribonucleotide reductase (RNR) are detected at DNA damage sites for local dNDP formation. Because deoxyuridine diphosphate is synthesized by RNR, the simultaneous recruitment of TMPK and RNR to DNA damage sites is critical for preventing deoxyuridine triphosphate-mediated toxic repair. This study investigates the mechanism responsible for the recruitment of TMPK to DNA damage sites. Our data demonstrate the requirement of ataxia telangiectasia mutated (ATM) kinase activity for TMPK recruitment to DNA lesion sites. Moreover, we find that TMPK is able to form the complex with histone acetyltransferase Tip60 and RNR. Inhibition of ATM kinase reduces the complex formation and TMPK phosphorylation. Our analysis further shows the presence of TMPK phosphorylation at serine 88, which is an ATM kinase consensus site. A phosphorylation-defective mutation at this site suppresses TMPK recruitment to DNA damage sites and the complex formation with Tip60. Finally, we provide evidence that this site is critical for the function of TMPK in DNA repair but not for catalytic activity. Together, these findings suggest that Tip60-ATM signaling has a functional contribution to the recruitment of TMPK to DNA damage sites, thereby increasing local dTTP synthesis for DNA repair.-Hu, C.-M., Tsao, N., Wang, Y.-T., Chen, Y.-J., Chang, Z.-F. Thymidylate kinase is critical for DNA repair via ATM-dependent Tip60 complex formation.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Dano ao DNA , Reparo do DNA , Lisina Acetiltransferase 5/metabolismo , Complexos Multienzimáticos/metabolismo , Núcleosídeo-Fosfato Quinase/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Células HEK293 , Células HeLa , Humanos , Lisina Acetiltransferase 5/genética , Complexos Multienzimáticos/genética , Núcleosídeo-Fosfato Quinase/genética , Fosforilação/genética , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismo
14.
J Exp Bot ; 70(4): 1167-1182, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30534992

RESUMO

The activity of ribonucleotide reductase (RNR), which catalyses the transformation of four ribonucleoside diphosphates (NDPs) to their corresponding deoxyribonucleoside diphosphates (dNDPs), is the main determiner of the cellular concentration of dNTP pools and should be tightly coordinated with DNA synthesis and cell-cycle progression. Constitutively increased or decreased RNR activity interferes with DNA replication and leads to arrested cell cycle progression; however, the mechanisms underlying these disruptive effects in higher plants remain to be uncovered. In this study, we identified a RNR large subunit mutant, sistl1, in Setaria italica (foxtail millet), which exhibited growth retardation as well as striped leaf phenotype, i.e. irregularly reduced leaf vein distances and decreased chloroplast biogenesis. We determined that a Gly737 to Glu substitution occurring in the C-terminus of the SiSTL1 protein slightly affected its optimal function, leading in turn to the reduced expression of genes variously involved in the assembly and activation of the DNA pre-replicative complex, elongation of replication forks and S phase entry. Our study provides new insights into how SiSTL1 regulates plant growth, chloroplast biogenesis, and cell cycle progression in Poaceae crops.


Assuntos
Ciclo Celular/fisiologia , Cloroplastos/fisiologia , Organogênese Vegetal , Proteínas de Plantas/genética , Ribonucleotídeo Redutases/genética , Setaria (Planta)/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Ribonucleotídeo Redutases/metabolismo , Alinhamento de Sequência , Setaria (Planta)/crescimento & desenvolvimento
15.
FEBS J ; 286(6): 1154-1173, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30536627

RESUMO

Chromatin regulates gene expression and genome maintenance, and consists of histones and other components. The post-translational modification of histones plays a key role in maintaining the structure and function of chromatin under different pathophysiological stress conditions. Here, we investigate the functions of previously unexplored amino acid residues in histones H3 and H4. To do so, we screened a library of yeast histone mutants following DNA damage and identified that substitution mutations of histone H3 (H3Q5A/E and H3Q120A) and H4 (H4Y88A/E and H4R78K) render yeast cells sensitive to DNA-damaging agents. These histone mutants show an activated DNA damage response, Rad53 phosphorylation and Sml1 degradation in the presence of methyl methanesulfonate (MMS). In histone H3Q5A/E mutants, RNR2 and RNR3 genes were induced at low level, as was RNR3 in H4 histone mutants following DNA damage. In H3 mutant cells, the cell cycle was deregulated, leading to inefficient cell cycle arrest in the presence of MMS, and genes involved in aging and DNA damage repair pathways were constitutively upregulated. In H3 mutants (H3Q5A, H3Q5E and H3Q120A), we observed reduced chronological lifespan (CLS), compared with extended CLS in the H4R78K mutant. Histone mutants also showed altered H3K4me and H3K56ac modifications and improper activation of the stress responsive Slt2 and Hog1 kinases. Thus, we have determined the significance of previously uncharacterized residues of H3 and H4 in DNA damage response, cell cycle progression and cellular aging.


Assuntos
Aminoácidos/genética , Senescência Celular , Dano ao DNA , Reparo do DNA , Histonas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Aminoácidos/química , Aminoácidos/metabolismo , Pontos de Checagem do Ciclo Celular , Cromatina , Histonas/metabolismo , Metanossulfonato de Metila/efeitos adversos , Mutação , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
16.
Curr Genet ; 65(2): 477-482, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30519713

RESUMO

The molecular chaperones Hsp70 and Hsp90 bind and fold a significant proportion of the proteome. They are responsible for the activity and stability of many disease-related proteins including those in cancer. Substantial effort has been devoted to developing a range of chaperone inhibitors for clinical use. Recent studies have identified the oncogenic ribonucleotide reductase (RNR) complex as an interactor of chaperones. While several generations of RNR inhibitor have been developed for use in cancer patients, many of these produce severe side effects such as nausea, vomiting and hair loss. Development of more potent, less patient-toxic anti-RNR strategies would be highly desirable. Inhibition of chaperones and associated co-chaperone molecules in both cancer and model organisms such as budding yeast result in the destabilization of RNR subunits and a corresponding sensitization to RNR inhibitors. Going forward, this may form part of a novel strategy to target cancer cells that are resistant to standard RNR inhibitors.


Assuntos
Chaperonas Moleculares/metabolismo , Ribonucleotídeo Redutases/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Dano ao DNA , Replicação do DNA , Ativação Enzimática , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/etiologia , Neoplasias/metabolismo , Ligação Proteica , Proteômica/métodos , Ribonucleotídeo Redutases/antagonistas & inibidores , Ribonucleotídeo Redutases/metabolismo
17.
Eur J Med Genet ; 62(11): 103574, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30439532

RESUMO

RRM2B encodes the crucial p53-inducible ribonucleotide reductase small subunit 2 homolog (p53R2), which is required for DNA synthesis throughout the cell cycle. Mutations in this gene have been associated with a lethal mitochondrial depletion syndrome. Here we present the case of an infant with a novel homozygous p.Asn221Ser mutation in RRM2B who developed hypotonia, failure to thrive, sensorineural hearing loss, and severe metabolic lactic acidosis, ultimately progressing to death at 3 months of age. Through molecular modeling using the X-ray crystal structure of p53R2, we demonstrate that this mutation likely causes disruption of a highly conserved helix region of the protein by altering intramolecular interactions. This report expands our knowledge of potential pathogenic RRM2B mutations as well as our understanding of the molecular function of p53R2 and its role in the pathogenesis of mitochondrial DNA depletion.


Assuntos
Acidose/genética , Proteínas de Ciclo Celular/genética , Morte Perinatal , Ribonucleotídeo Redutases/genética , Acidose/diagnóstico por imagem , Acidose/patologia , Proteínas de Ciclo Celular/química , Cristalografia por Raios X , Feminino , Homozigoto , Humanos , Lactente , Recém-Nascido , Masculino , Mutação/genética , Gravidez , Conformação Proteica , Ribonucleotídeo Redutases/química
18.
Nat Microbiol ; 4(1): 78-88, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30420783

RESUMO

The apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide-like (APOBEC) family of single-stranded DNA (ssDNA) cytosine deaminases provides innate immunity against virus and transposon replication1-4. A well-studied mechanism is APOBEC3G restriction of human immunodeficiency virus type 1, which is counteracted by a virus-encoded degradation mechanism1-4. Accordingly, most work has focused on retroviruses with obligate ssDNA replication intermediates and it is unclear whether large double-stranded DNA (dsDNA) viruses may be similarly susceptible to restriction. Here, we show that the large dsDNA herpesvirus Epstein-Barr virus (EBV), which is the causative agent of infectious mononucleosis and multiple cancers5, utilizes a two-pronged approach to counteract restriction by APOBEC3B. Proteomics studies and immunoprecipitation experiments showed that the ribonucleotide reductase large subunit of EBV, BORF26,7, binds APOBEC3B. Mutagenesis mapped the interaction to the APOBEC3B catalytic domain, and biochemical studies demonstrated that BORF2 stoichiometrically inhibits APOBEC3B DNA cytosine deaminase activity. BORF2 also caused a dramatic relocalization of nuclear APOBEC3B to perinuclear bodies. On lytic reactivation, BORF2-null viruses were susceptible to APOBEC3B-mediated deamination as evidenced by lower viral titres, lower infectivity and hypermutation. The Kaposi's sarcoma-associated herpesvirus homologue, ORF61, also bound APOBEC3B and mediated relocalization. These data support a model where the genomic integrity of human γ-herpesviruses is maintained by active neutralization of the antiviral enzyme APOBEC3B.


Assuntos
Citidina Desaminase/antagonistas & inibidores , Herpesvirus Humano 4/metabolismo , Herpesvirus Humano 8/metabolismo , Ribonucleotídeo Redutases/metabolismo , Proteínas Virais/metabolismo , Sistemas CRISPR-Cas , Domínio Catalítico/genética , Linhagem Celular , Genoma Viral/genética , Células HEK293 , Herpesvirus Humano 4/crescimento & desenvolvimento , Humanos , Antígenos de Histocompatibilidade Menor , Interferência de RNA , RNA Interferente Pequeno/genética , Ribonucleotídeo Redutases/genética , Proteínas Virais/genética
19.
PLoS One ; 13(10): e0204111, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30273369

RESUMO

BACKGROUND: The use of the HIV antiretroviral drug stavudine (d4T), a thymidine analogue, is associated with the development of mitochondrial toxicities such as sensory neuropathy (SN). Genetic variation in genes relating to d4T transport and metabolism, as well as genetic variation in the thymidine synthesis pathway, could influence occurrence of d4T-related toxicity. METHODS: We examined this hypothesis in a cohort of HIV-positive South African adults exposed to d4T, including 143 cases with SN and 120 controls without SN. Ten SNPs in four genes associated with stavudine transport, and 16 SNPs in seven genes of the thymidine synthesis / phosphorylation pathway were genotyped using Agena mass spectrometry methods. Associations between sensory neuropathy and genetic variants were evaluated using PLINK by univariate and multivariable analyses. RESULTS: Age and height were significantly associated with SN occurrence. Using logistic regression with age and height as covariates, and uncorrected empirical p-values, genetic variation in SLC28A1, SAMHD1, MTHFR and RRM2B was associated with SN in South Africans using d4T. CONCLUSION: Variation in genes relating to d4T transport and metabolism, as well as genetic variation in the thymidine synthesis pathway may influence occurrence of d4T-related SN. These data contribute to the characterisation of African pharmacogenetic variation and its role in adverse response to antiretroviral therapy.


Assuntos
Fármacos Anti-HIV/efeitos adversos , Infecções por HIV/tratamento farmacológico , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Variantes Farmacogenômicos , Polimorfismo de Nucleotídeo Único , Estavudina/efeitos adversos , Adulto , Grupo com Ancestrais do Continente Africano , Fármacos Anti-HIV/uso terapêutico , Estudos de Casos e Controles , Proteínas de Ciclo Celular/genética , Feminino , Infecções por HIV/genética , Humanos , Masculino , Proteínas de Membrana Transportadoras/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Pessoa de Meia-Idade , Doenças do Sistema Nervoso Periférico/genética , Ribonucleotídeo Redutases/genética , Proteína 1 com Domínio SAM e Domínio HD/genética , África do Sul , Estavudina/uso terapêutico
20.
J Biol Chem ; 293(41): 15889-15900, 2018 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-30166338

RESUMO

Class I ribonucleotide reductase (RNR) consists of a catalytic subunit (NrdA) and a radical-generating subunit (NrdB) that together catalyze reduction of ribonucleotides to their corresponding deoxyribonucleotides. NrdB from the firmicute Facklamia ignava is a unique fusion protein with N-terminal add-ons of a glutaredoxin (Grx) domain followed by an ATP-binding domain, the ATP cone. Grx, usually encoded separately from the RNR operon, is a known RNR reductant. We show that the fused Grx domain functions as an efficient reductant of the F. ignava class I RNR via the common dithiol mechanism and, interestingly, also via a monothiol mechanism, although less efficiently. To our knowledge, a Grx that uses both of these two reaction mechanisms has not previously been observed with a native substrate. The ATP cone is in most RNRs an N-terminal domain of the catalytic subunit. It is an allosteric on/off switch promoting ribonucleotide reduction in the presence of ATP and inhibiting RNR activity in the presence of dATP. We found that dATP bound to the ATP cone of F. ignava NrdB promotes formation of tetramers that cannot form active complexes with NrdA. The ATP cone bound two dATP molecules but only one ATP molecule. F. ignava NrdB contains the recently identified radical-generating cofactor MnIII/MnIV We show that NrdA from F. ignava can form a catalytically competent RNR with the MnIII/MnIV-containing NrdB from the flavobacterium Leeuwenhoekiella blandensis In conclusion, F. ignava NrdB is fused with a Grx functioning as an RNR reductant and an ATP cone serving as an on/off switch.


Assuntos
Glutarredoxinas/metabolismo , Ribonucleotídeo Redutases/metabolismo , Aerococcaceae/química , Catálise , Nucleotídeos de Desoxiadenina/metabolismo , Flavobacteriaceae/química , Transferência Genética Horizontal , Glutarredoxinas/química , Glutarredoxinas/genética , Oxirredução , Ligação Proteica , Domínios Proteicos , Multimerização Proteica/efeitos dos fármacos , Ribonucleotídeo Redutases/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA