Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.358
Filtrar
1.
Nucleic Acids Res ; 49(4): 2179-2191, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33533925

RESUMO

Replication forks often stall at damaged DNA. To overcome these obstructions and complete the DNA duplication in a timely fashion, replication can be restarted downstream of the DNA lesion. In mammalian cells, this repriming of replication can be achieved through the activities of primase and polymerase PrimPol. PrimPol is stimulated in DNA synthesis through interaction with PolDIP2, however the exact mechanism of this PolDIP2-dependent stimulation is still unclear. Here, we show that PrimPol uses a flexible loop to interact with the C-terminal ApaG-like domain of PolDIP2, and that this contact is essential for PrimPol's enhanced processivity. PolDIP2 increases primer-template and dNTP binding affinities of PrimPol, which concomitantly enhances its nucleotide incorporation efficiency. This stimulation is dependent on a unique arginine cluster in PolDIP2. Since the polymerase activity of PrimPol alone is very limited, this mechanism, where the affinity for dNTPs gets increased by PolDIP2 binding, might be critical for the in vivo function of PrimPol in tolerating DNA lesions at physiological nucleotide concentrations.


Assuntos
Arginina/química , DNA Primase/química , DNA Polimerase Dirigida por DNA/química , DNA/biossíntese , Enzimas Multifuncionais/química , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Motivos de Aminoácidos , DNA Primase/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/metabolismo , Modelos Moleculares , Enzimas Multifuncionais/metabolismo , Ligação Proteica
2.
Nat Commun ; 12(1): 796, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542236

RESUMO

RNA polymerases (RNAPs) synthesize RNA from NTPs, whereas DNA polymerases synthesize DNA from 2'dNTPs. DNA polymerases select against NTPs by using steric gates to exclude the 2'OH, but RNAPs have to employ alternative selection strategies. In single-subunit RNAPs, a conserved Tyr residue discriminates against 2'dNTPs, whereas selectivity mechanisms of multi-subunit RNAPs remain hitherto unknown. Here, we show that a conserved Arg residue uses a two-pronged strategy to select against 2'dNTPs in multi-subunit RNAPs. The conserved Arg interacts with the 2'OH group to promote NTP binding, but selectively inhibits incorporation of 2'dNTPs by interacting with their 3'OH group to favor the catalytically-inert 2'-endo conformation of the deoxyribose moiety. This deformative action is an elegant example of an active selection against a substrate that is a substructure of the correct substrate. Our findings provide important insights into the evolutionary origins of biopolymers and the design of selective inhibitors of viral RNAPs.


Assuntos
Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Desoxirribonucleotídeos/metabolismo , Desoxirribose/metabolismo , Arginina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/ultraestrutura , Cristalografia por Raios X , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/isolamento & purificação , RNA Polimerases Dirigidas por DNA/ultraestrutura , Escherichia coli/enzimologia , Escherichia coli/genética , Cinética , Simulação de Acoplamento Molecular , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Especificidade por Substrato , Thermus thermophilus/enzimologia , Thermus thermophilus/genética
3.
Nucleic Acids Res ; 49(5): 2598-2608, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33591315

RESUMO

Aberrant end joining of DNA double strand breaks leads to chromosomal rearrangements and to insertion of nuclear or mitochondrial DNA into breakpoints, which is commonly observed in cancer cells and constitutes a major threat to genome integrity. However, the mechanisms that are causative for these insertions are largely unknown. By monitoring end joining of different linear DNA substrates introduced into HEK293 cells, as well as by examining end joining of CRISPR/Cas9 induced DNA breaks in HEK293 and HeLa cells, we provide evidence that the dNTPase activity of SAMHD1 impedes aberrant DNA resynthesis at DNA breaks during DNA end joining. Hence, SAMHD1 expression or low intracellular dNTP levels lead to shorter repair joints and impede insertion of distant DNA regions prior end repair. Our results reveal a novel role for SAMHD1 in DNA end joining and provide new insights into how loss of SAMHD1 may contribute to genome instability and cancer development.


Assuntos
Reparo do DNA por Junção de Extremidades , Proteína 1 com Domínio SAM e Domínio HD/fisiologia , Proteína 9 Associada à CRISPR/metabolismo , Quebra Cromossômica , Desoxirribonucleotídeos/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteína 1 com Domínio SAM e Domínio HD/metabolismo
4.
Mol Cell ; 78(3): 396-410.e4, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32169162

RESUMO

The Mec1 and Rad53 kinases play a central role during acute replication stress in budding yeast. They are also essential for viability in normal growth conditions, but the signal that activates the Mec1-Rad53 pathway in the absence of exogenous insults is currently unknown. Here, we show that this pathway is active at the onset of normal S phase because deoxyribonucleotide triphosphate (dNTP) levels present in G1 phase may not be sufficient to support processive DNA synthesis and impede DNA replication. This activation can be suppressed experimentally by increasing dNTP levels in G1 phase. Moreover, we show that unchallenged cells entering S phase in the absence of Rad53 undergo irreversible fork collapse and mitotic catastrophe. Together, these data indicate that cells use suboptimal dNTP pools to detect the onset of DNA replication and activate the Mec1-Rad53 pathway, which in turn maintains functional forks and triggers dNTP synthesis, allowing the completion of DNA replication.


Assuntos
Replicação do DNA/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fase S/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Desoxirribonucleotídeos/genética , Desoxirribonucleotídeos/metabolismo , Regulação Fúngica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mitose , Proteínas Serina-Treonina Quinases/genética , Origem de Replicação , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/genética
5.
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
6.
J Biol Chem ; 295(2): 657-666, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31806704

RESUMO

Unlike activated CD4+ T cells, nondividing macrophages have an extremely small dNTP pool, which restricts HIV-1 reverse transcription. However, rNTPs are equally abundant in both of these cell types and reach much higher concentrations than dNTPs. The greater difference in concentration between dNTPs and rNTPs in macrophages results in frequent misincorporation of noncanonical rNTPs during HIV-1 reverse transcription. Here, we tested whether the highly abundant SAM domain- and HD domain-containing protein 1 (SAMHD1) deoxynucleoside triphosphorylase in macrophages is responsible for frequent rNTP incorporation during HIV-1 reverse transcription. We also assessed whether Vpx (viral protein X), an accessory protein of HIV-2 and some simian immunodeficiency virus strains that targets SAMHD1 for proteolytic degradation, can counteract the rNTP incorporation. Results from biochemical simulation of HIV-1 reverse transcriptase-mediated DNA synthesis confirmed that rNTP incorporation is reduced under Vpx-mediated dNTP elevation. Using HIV-1 vector, we further demonstrated that dNTP pool elevation by Vpx or deoxynucleosides in human primary monocyte-derived macrophages reduces noncanonical rNTP incorporation during HIV-1 reverse transcription, an outcome similarly observed with the infectious HIV-1 89.6 strain. Furthermore, the simian immunodeficiency virus mac239 strain, encoding Vpx, displayed a much lower level of rNTP incorporation than its ΔVpx mutant in macrophages. Finally, the amount of rNMPs incorporated in HIV-1 proviral DNAs remained unchanged for ∼2 weeks in macrophages. These findings suggest that noncanonical rNTP incorporation is regulated by SAMHD1 in macrophages, whereas rNMPs incorporated in HIV-1 proviral DNA remain unrepaired. This suggests a potential long-term DNA damage impact of SAMHD1-mediated rNTP incorporation in macrophages.


Assuntos
Infecções por HIV/metabolismo , HIV/metabolismo , Macrófagos/virologia , Transcrição Reversa , Ribonucleotídeos/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Células Cultivadas , Desoxirribonucleotídeos/genética , Desoxirribonucleotídeos/metabolismo , HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/genética , HIV-1/metabolismo , HIV-2/genética , HIV-2/metabolismo , Humanos , Células Jurkat , Macrófagos/metabolismo , Mutagênese , Ribonucleotídeos/genética , Proteína 1 com Domínio SAM e Domínio HD/metabolismo
7.
DNA Cell Biol ; 39(1): 50-56, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31750734

RESUMO

POLD1 encodes the catalytic subunit of DNA polymerase delta (Polδ), the major lagging strand polymerase, which also participates in DNA repair. Mutations affecting the exonuclease domain increase the risk of various cancers, while mutations that change the polymerase active site cause a progeroid syndrome called mandibular hypoplasia, deafness, progeroid features, and lipodystrophy (MDPL) syndrome. We generated a set of catalytic subunit of human telomerase (hTERT)-immortalized human fibroblasts expressing wild-type or mutant POLD1 using the retroviral LXSN vector system. In the resulting cell lines, expression of endogenous POLD1 was suppressed in favor of the recombinant POLD1. The siRNA screening of DNA damage-related genes revealed that fibroblasts expressing D316H and S605del POLD1 were more sensitive to knockdowns of ribonuclease reductase (RNR) components, RRM1 and RRM2 in the presence of hydroxyurea (HU), an RNR inhibitor. On the contrary, SAMHD1 siRNA, which increases the concentration of dNTPs, increased growth of wild type, D316H, and S605del POLD1 fibroblasts. Hypersensitivity to dNTP synthesis inhibition in POLD1 mutant lines was confirmed using gemcitabine. Our finding is consistent with the notion that reduced dNTP concentration negatively affects the cell growth of hTERT fibroblasts expressing exonuclease and polymerase mutant POLD1.


Assuntos
DNA Polimerase III/genética , Desoxirribonucleotídeos/metabolismo , Fibroblastos/metabolismo , Mutação , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , DNA Polimerase III/antagonistas & inibidores , DNA Polimerase III/metabolismo , Surdez/genética , Surdez/metabolismo , Exonucleases/genética , Exonucleases/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Hidroxiureia/farmacologia , Lipodistrofia/genética , Lipodistrofia/metabolismo , Lipodistrofia/patologia , Interferência de RNA , Proteína 1 com Domínio SAM e Domínio HD/genética , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Síndrome
8.
BMC Med Res Methodol ; 19(1): 216, 2019 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-31775643

RESUMO

BACKGROUND: Antiretroviral therapy (ART) has significantly reduced HIV-related morbidity and mortality. However, therapeutic benefit of ART is often limited by delayed drug-associated toxicity. Nucleoside reverse transcriptase inhibitors (NRTIs) are the backbone of ART regimens. NRTIs compete with endogenous deoxyribonucleotide triphosphates (dNTPs) in incorporation into elongating DNA chain resulting in their cytotoxic or antiviral effect. Thus, the efficacy of NRTIs could be affected by direct competition with endogenous dNTPs and/or feedback inhibition of their metabolic enzymes. In this paper, we assessed whether the levels of ribonucleotides (RN) and dNTP pool sizes can be used as biomarkers in distinguishing between HIV-infected patients with ART-induced mitochondrial toxicity and HIV-infected patients without toxicity. METHODS: We used data collected through a case-control study from 50 subjects. Cases were defined as HIV-infected individuals with clinical and/or laboratory evidence of mitochondrial toxicity. Each case was age, gender, and race matched with an HIV-positive without evidence of toxicity. We used a range of machine learning procedures to distinguish between patients with and without toxicity. Using resampling methods like Monte Carlo k-fold cross validation, we compared the accuracy of several machine learning algorithms applied to our data. We used the algorithm with highest classification accuracy rate in evaluating the diagnostic performance of 12 RN and 14 dNTP pool sizes as biomarkers of mitochondrial toxicity. RESULTS: We used eight classification algorithms to assess the diagnostic performance of RN and dNTP pool sizes distinguishing HIV patients with and without NRTI-associated mitochondrial toxicity. The algorithms resulted in cross-validated classification rates of 0.65-0.76 for dNTP and 0.72-0.83 for RN, following reduction of the dimensionality of the input data. The reduction of input variables improved the classification performance of the algorithms, with the most pronounced improvement for RN. Complex tree-based methods worked the best for both the deoxyribose dataset (Random Forest) and the ribose dataset (Classification Tree and AdaBoost), but it is worth noting that simple methods such as Linear Discriminant Analysis and Logistic Regression were very competitive in terms of classification performance. CONCLUSIONS: Our finding of changes in RN and dNTP pools in participants with mitochondrial toxicity validates the importance of dNTP pools in mitochondrial function. Hence, levels of RN and dNTP pools can be used as biomarkers of ART-induced mitochondrial toxicity.


Assuntos
Antirretrovirais/efeitos adversos , Desoxirribonucleotídeos/metabolismo , Didesoxinucleotídeos/metabolismo , Infecções por HIV/tratamento farmacológico , Aprendizado de Máquina , Ribonucleotídeos/metabolismo , Algoritmos , Biomarcadores/metabolismo , Estudos de Casos e Controles , Infecções por HIV/diagnóstico , Humanos
10.
Postepy Biochem ; 65(2): 143-152, 2019 06 06.
Artigo em Polonês | MEDLINE | ID: mdl-31642653

RESUMO

High replication fidelity, understood as the DNA polymerases' ability to select nucleotides with both correct base and sugar, is of critical importance for maintaining the genetic stability. Due to the fact that the cellular levels of ribonucleotides are much higher than the concentrations of deoxyribonucleotides, replicative polymerases are able to incorporate ribonucleotides with up to 1000-fold higher frequency than mismatched deoxyribonucleotides. The ability to discriminate against ribonucleotides by the DNA polymerases relies on the steric gate residue in the enzyme's catalytic centre. Despite the fact that ribonucleotides are the most abundantly inserted incorrect nucleotides in DNA, they are not observed in properly functioning cells. The major pathway responsible for the recognition and removal of ribonucleotides from DNA is called Ribonucleotide Excision Repair. The impairment of ribonucleotide removal pathways can cause increased mutation rate, replication stress, DNA breakage, problems with transcription, chromatin structure maintenance, genetic disorders and cell death. In spite of that, ribonucleotide incorporation into DNA may have some positive biological impact, stimulating mismatch repair and non-homologous end joining.


Assuntos
Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/química , DNA/metabolismo , Instabilidade Genômica , Ribonucleotídeos/metabolismo , DNA/genética , Replicação do DNA , Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/metabolismo , Ribonucleotídeos/química
11.
Chin J Nat Med ; 17(8): 608-615, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31472898

RESUMO

In an effort to understand the molecular events contributing to the cytotoxicity activity of resveratrol (RSV), we investigated its effects on human lung adenocarcinoma epithelial cell line A549 at different concentrations. Cellular nucleoside metabolic profiling was determined by an established liquid chromatography-mass spectrometry method in A549 cells. RSV resulted in significant decreases and imbalances of deoxyribonucleoside triphosphates (dNTPs) pools suppressing subsequent DNA synthesis. Meanwhile, RSV at high concentration caused significant cell cycle arrest at S phase, in which cells required the highest dNTPs supply than other phases for DNA replication. The inhibition of DNA synthesis thus blocked subsequent progression through S phase in A549 cells, which may partly contribute to the cytotoxicity effect of RSV. However, hydroxyurea (HU), an inhibitor of RNR activity, caused similar dNTPs perturbation but no S phase arrest, finally no cytotoxicity effect. Therefore, we believed that the dual effect of high concentration RSV, including S phase arrest and DNA synthesis inhibition, was required for its cytotoxicity effect on A549 cells. In summary, our results provided important clues to the molecular basis for the anticancer effect of RSV on epithelial cells.


Assuntos
Adenocarcinoma de Pulmão/patologia , Ciclo Celular/efeitos dos fármacos , Desoxirribonucleotídeos/metabolismo , Células Epiteliais/efeitos dos fármacos , Neoplasias Pulmonares/patologia , Resveratrol/farmacologia , Células A549 , Adenocarcinoma de Pulmão/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Hidroxiureia/farmacologia , Neoplasias Pulmonares/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos
12.
Virology ; 537: 36-44, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31442614

RESUMO

Unlike HIV-1, HIV-2 and some SIV strains replicate at high dNTP concentrations even in macrophages due to their accessory proteins, Vpx or Vpr, that target SAMHD1 dNTPase for proteasomal degradation. We previously reported that HIV-1 reverse transcriptase (RT) efficiently synthesizes DNA even at low dNTP concentrations because HIV-1 RT displays faster pre-steady state kpol values than SAMHD1 counteracting lentiviral RTs. Here, since the kpol step consists of two sequential sub-steps post dNTP binding, conformational change and chemistry, we investigated which of the two sub-steps RTs from SAMHD1 non-counteracting viruses accelerate in order to complete reverse transcription in the limited dNTP pools found in macrophages. Our study demonstrates that RTs of SAMHD1 non-counteracting lentiviruses have a faster conformational change rate during dNTP incorporation, supporting that these lentiviruses may have evolved to harbor RTs that can efficiently execute the conformational change step in order to circumvent SAMHD1 restriction and dNTP depletion in macrophages.


Assuntos
Desoxirribonucleotídeos/metabolismo , Lentivirus de Primatas/enzimologia , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/metabolismo , Animais , Linhagem Celular , Humanos , Cinética , Primatas , Conformação Proteica
13.
Anal Chem ; 91(16): 10381-10385, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31364352

RESUMO

DNA damage seriously threats the genomic stability and is linked to mutagenesis, carcinogenesis, and cell death. DNA damage includes the isolated damage and the clustered damages, but few approaches are available for efficient detection of the clustered damage due to its spatial distribution. Herein, we present a single-molecule counting approach with the capability of detecting both the isolated and the clustered damages in genomic DNAs. We employed the repair enzymes to remove the DNA damage and used the terminal deoxynucleotidyl transferase (TdT) to incorporate biotinylated nucleotides and fluorescent nucleotides into the damage sites in a template-independent manner. The number of total oxidative damaged bases is quantified to be 7328-7406 in a single HeLa cell treated with 150 µM H2O2. This method in combination with special repair enzymes can detect a variety of DNA damage in different types of cells, holding great potential for early diagnosis of DNA damage-related human diseases.


Assuntos
Reparo do DNA/efeitos dos fármacos , DNA/análise , Imagem Individual de Molécula/métodos , Coloração e Rotulagem/métodos , Biotina/química , Biotinilação , Carbocianinas/química , DNA/genética , DNA/metabolismo , Dano ao DNA , DNA Glicosilases/química , DNA Glicosilases/metabolismo , DNA Nucleotidilexotransferase/química , DNA Nucleotidilexotransferase/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/metabolismo , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Estreptavidina/química
14.
FEBS J ; 286(19): 3844-3857, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31152619

RESUMO

Sterile α-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) is an intrinsic antiviral restriction factor known to play a vital role in preventing multiple viral infections and in the control of the cellular deoxynucleoside triphosphate (dNTP) pool. Human and mouse SAMHD1 have both been extensively studied; however, our knowledge on porcine SAMHD1 is limited. Here, we report our findings from comprehensive structural and functional studies on porcine SAMHD1. We determined the crystal structure of porcine SAMHD1 and showed that it forms a symmetric tetramer. Moreover, we modified the deoxynucleotide triphosphohydrolase (dNTPase) activity of SAMHD1 by site-directed mutagenesis based on the crystal structure, and obtained an artificial dimeric enzyme possessing high dNTPase activity. Taken together, our results define the mechanism underlying dNTP regulation and provide a deeper understanding of the regulation of porcine SAMHD1 functions. Directed modification of key residues based on the protein structure enhances the activity of the enzyme, which will be beneficial in the search for new antiviral strategies and for future translational applications.


Assuntos
Desoxirribonucleotídeos/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/química , Animais , Biopolímeros/química , Cristalografia por Raios X , Guanosina Trifosfato/química , Conformação Proteica , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Suínos
15.
Curr Biol ; 29(7): 1187-1192.e3, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30880011

RESUMO

The egg contains maternal RNAs and proteins, which have instrumental functions in patterning and morphogenesis. Besides these, the egg also contains metabolites, whose developmental functions have been little investigated. For example, the rapid increase of DNA content during the fast embryonic cell cycles poses high demands on the supply of deoxyribonucleotides (dNTPs), which may be synthesized in the embryo or maternally provided [1, 2]. Here, we analyze the role of dNTP in early Drosophila embryos. We found that dNTP levels initially decreased about 2-fold before reaching stable levels at the transition from syncytial to cellular blastoderm. Employing a mutant of the metabolic enzyme serine hydroxymethyl transferase (SHMT), which is impaired in the embryonic synthesis of deoxythymidine triphosphate (dTTP), we found that the maternal supply of dTTP was specifically depleted by interphase 13. SHMT mutants showed persistent S phase, replication stress, and a checkpoint-dependent cell-cycle arrest in NC13, depending on the loss of dTTP. The cell-cycle arrest in SHMT mutants was suppressed by reduced zygotic transcription. Consistent with the requirement of dTTP for cell-cycle progression, increased dNTP levels accelerated the cell cycle in embryos lacking zygotic transcription. We propose a model that both a limiting dNTP supply and interference of zygotic transcription with DNA replication [3] elicit DNA replication stress and checkpoint activation. Our study reveals a specific mechanism of how dNTP metabolites contribute to the embryonic cell-cycle control.


Assuntos
Pontos de Checagem do Ciclo Celular , Desoxirribonucleotídeos/metabolismo , Drosophila/fisiologia , Animais , Drosophila/embriologia , Embrião não Mamífero/fisiologia
16.
FASEB J ; 33(6): 7168-7179, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30848931

RESUMO

Polymerase γ catalytic subunit (POLG) gene encodes the enzyme responsible for mitochondrial DNA (mtDNA) synthesis. Mutations affecting POLG are the most prevalent cause of mitochondrial disease because of defective mtDNA replication and lead to a wide spectrum of clinical phenotypes characterized by mtDNA deletions or depletion. Enhancing mitochondrial deoxyribonucleoside triphosphate (dNTP) synthesis effectively rescues mtDNA depletion in different models of defective mtDNA maintenance due to dNTP insufficiency. In this study, we studied mtDNA copy number recovery rates following ethidium bromide-forced depletion in quiescent fibroblasts from patients harboring mutations in different domains of POLG. Whereas control cells spontaneously recovered initial mtDNA levels, POLG-deficient cells experienced a more severe depletion and could not repopulate mtDNA. However, activation of deoxyribonucleoside (dN) salvage by supplementation with dNs plus erythro-9-(2-hydroxy-3-nonyl) adenine (inhibitor of deoxyadenosine degradation) led to increased mitochondrial dNTP pools and promoted mtDNA repopulation in all tested POLG-mutant cells independently of their specific genetic defect. The treatment did not compromise POLG fidelity because no increase in multiple deletions or point mutations was detected. Our study suggests that physiologic dNTP concentration limits the mtDNA replication rate. We thus propose that increasing mitochondrial dNTP availability could be of therapeutic interest for POLG deficiency and other conditions in which mtDNA maintenance is challenged.-Blázquez-Bermejo, C., Carreño-Gago, L., Molina-Granada, D., Aguirre, J., Ramón, J., Torres-Torronteras, J., Cabrera-Pérez, R., Martín, M. Á., Domínguez-González, C., de la Cruz, X., Lombès, A., García-Arumí, E., Martí, R., Cámara, Y. Increased dNTP pools rescue mtDNA depletion in human POLG-deficient fibroblasts.


Assuntos
Polimerase do DNA Mitocondrial/deficiência , DNA Mitocondrial/metabolismo , Desoxirribonucleotídeos/farmacologia , Fibroblastos/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Adulto , Domínio Catalítico/genética , Células Cultivadas , Polimerase do DNA Mitocondrial/genética , Replicação do DNA/efeitos dos fármacos , DNA Mitocondrial/genética , Desoxirribonucleotídeos/metabolismo , Etídio/farmacologia , Feminino , Fibroblastos/efeitos dos fármacos , Genótipo , Humanos , Masculino , Mitocôndrias Musculares/genética , Modelos Moleculares , Mutação de Sentido Incorreto , Fenótipo , Mutação Puntual , Conformação Proteica , Reação em Cadeia da Polimerase em Tempo Real , Deleção de Sequência
17.
Environ Microbiol Rep ; 11(3): 448-455, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30809954

RESUMO

Bacteriophage possess a variety of auxiliary metabolic genes of bacterial origin. These proteins enable them to maximize infection efficiency, subverting bacterial metabolic processes for the purpose of viral genome replication and synthesis of the next generation of virion progeny. Here, we examined the enzymatic activity of a cyanophage MazG protein - a putative pyrophosphohydrolase previously implicated in regulation of the stringent response via reducing levels of the central alarmone molecule (p)ppGpp. We demonstrate, however, that the purified viral MazG shows no binding or hydrolysis activity against (p)ppGpp. Instead, dGTP and dCTP appear to be the preferred substrates of this protein, consistent with a role preferentially hydrolysing deoxyribonucleotides from the high GC content host Synechococcus genome. This showcases a new example of the fine-tuned nature of viral metabolic processes.


Assuntos
Bacteriófagos/enzimologia , Desoxirribonucleotídeos/metabolismo , Pirofosfatases/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriófagos/classificação , Bacteriófagos/genética , Composição de Bases , Domínio Catalítico , Genoma Bacteriano/genética , Hidrólise , Filogenia , Pirofosfatases/química , Pirofosfatases/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Synechococcus/classificação , Synechococcus/enzimologia , Synechococcus/genética , Synechococcus/virologia , Proteínas Virais/química , Proteínas Virais/genética
18.
FASEB J ; 33(3): 4525-4537, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30702927

RESUMO

It has been shown that 5-amino-4-imidazolecarboxamide riboside (AICAr) can inhibit cell proliferation and induce apoptosis in childhood acute lymphoblastic leukemia (ALL) cells. Although AICAr could regulate cellular energy metabolism by activating AMPK, the cytotoxic mechanisms of AICAr are still unclear. Here, we knocked out TP53 or PRKAA1 gene (encoding AMPKα1) in NALM-6 and Reh cells by using the clustered regularly interspaced short palindromic repeats/Cas9 system and found that AICAr-induced proliferation inhibition was independent of AMPK activation but dependent on p53. Liquid chromatography-mass spectrometry analysis of nucleotide metabolites indicated that AICAr caused an increase in adenosine triphosphate, deoxyadenosine triphosphate, and deoxyguanosine triphosphate levels by up-regulating purine biosynthesis, while AICAr led to a decrease in cytidine triphosphate, uridine triphosphate, deoxycytidine triphosphate, and deoxythymidine triphosphate levels because of reduced phosphoribosyl pyrophosphate production, which consequently impaired the pyrimidine biosynthesis. Ribonucleoside triphosphate (NTP) pool imbalances suppressed the rRNA transcription efficiency. Furthermore, deoxy-ribonucleoside triphosphate (dNTP) pool imbalances induced DNA replication stress and DNA double-strand breaks, followed by cell cycle arrest and apoptosis in ALL cells. Exogenous uridine could rebalance the NTP and dNTP pools by supplementing pyrimidine and then attenuate AICAr-induced cytotoxicity. Our data indicate that RNA transcription inhibition and DNA replication stress induced by NTP and dNTP pool imbalances might play a key role in AICAr-mediated cytotoxic effects on ALL cells, suggesting a potential clinical application of AICAr in future ALL therapy.-Du, L., Yang, F., Fang, H., Sun, H., Chen, Y., Xu, Y., Li, H., Zheng, L., Zhou, B.-B. S. AICAr suppresses cell proliferation by inducing NTP and dNTP pool imbalances in acute lymphoblastic leukemia cells.


Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Nucleotídeos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Ribonucleotídeos/farmacologia , Proteínas Quinases Ativadas por AMP/deficiência , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/fisiologia , Aminoimidazol Carboxamida/antagonistas & inibidores , Aminoimidazol Carboxamida/farmacologia , Aminoimidazol Carboxamida/toxicidade , Apoptose/efeitos dos fármacos , Sistemas CRISPR-Cas , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Desoxirribonucleotídeos/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Técnicas de Inativação de Genes , Genes p53 , Genes de RNAr , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , RNA Ribossômico/biossíntese , Ribonucleotídeos/antagonistas & inibidores , Ribonucleotídeos/metabolismo , Ribonucleotídeos/toxicidade , Transcrição Genética/efeitos dos fármacos , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/fisiologia , Uridina/farmacologia
19.
Anal Biochem ; 568: 65-72, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30605633

RESUMO

Quantification of cellular deoxyribonucleoside mono- (dNMP), di- (dNDP), triphosphates (dNTPs) and related nucleoside metabolites are difficult due to their physiochemical properties and widely varying abundance. Involvement of dNTP metabolism in cellular processes including senescence and pathophysiological processes including cancer and viral infection make dNTP metabolism an important bioanalytical target. We modified a previously developed ion pairing reversed phase chromatography-mass spectrometry method for the simultaneous quantification and 13C isotope tracing of dNTP metabolites. dNMPs, dNDPs, and dNTPs were chromatographically resolved to avoid mis-annotation of in-source fragmentation. We used commercially available 13C15N-stable isotope labeled analogs as internal standards and show that this isotope dilution approach improves analytical figures of merit. At sufficiently high mass resolution achievable on an Orbitrap mass analyzer, stable isotope resolved metabolomics allows simultaneous isotope dilution quantification and 13C isotope tracing from major substrates including 13C-glucose. As a proof of principle, we quantified dNMP, dNDP and dNTP pools from multiple cell lines. We also identified isotopologue enrichment from glucose corresponding to ribose from the pentose-phosphate pathway in dNTP metabolites.


Assuntos
Desoxirribonucleotídeos/análise , Técnicas de Diluição do Indicador , Espectrometria de Massas , Isótopos de Carbono , Células Cultivadas , Cromatografia Líquida , Desoxirribonucleotídeos/metabolismo , Humanos , Marcação por Isótopo , Isótopos de Nitrogênio
20.
J Mol Biol ; 431(4): 673-686, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30633872

RESUMO

PrimPol is the most recently discovered human DNA polymerase/primase and plays an emerging role in nuclear and mitochondrial genomic maintenance. As a member of archaeo-eukaryotic primase superfamily enzymes, PrimPol possesses DNA polymerase and primase activities that are important for replication fork progression in vitro and in cellulo. The enzymatic activities of PrimPol are critically dependent on the nucleotidyl-transfer reaction to incorporate deoxyribonucleotides successively; however, our knowledge concerning the kinetic mechanism of the reaction remains incomplete. Using enzyme kinetic analyses and computer simulations, we dissected the mechanism by which PrimPol transfers a nucleotide to a primer-template DNA, which comprises DNA binding, conformational transition, nucleotide binding, phosphoester bond formation, and dissociation steps. We obtained the rate constants of the steps by steady-state and pre-steady-state kinetic analyses and simulations. Our data demonstrate that the rate-limiting step of PrimPol-catalyzed DNA elongation depends on the metal cofactor involved. In the presence of Mn2+, a conformational transition step from non-productive to productive PrimPol:DNA complexes limits the enzymatic turnover, whereas in the presence of Mg2+, the chemical step becomes rate limiting. As evidenced from our kinetic and simulation data, PrimPol maintains the same kinetic mechanism under either millimolar or physiological micromolar Mn2+ concentration. Our study revealed the underlying mechanism by which PrimPol catalyzes nucleotide incorporation with two common metal cofactors and provides a kinetic basis for further understanding the regulatory mechanism of this functionally diverse primase-polymerase.


Assuntos
Cátions Bivalentes/metabolismo , DNA Primase/metabolismo , DNA Catalítico/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Enzimas Multifuncionais/metabolismo , Catálise , Primers do DNA/genética , Replicação do DNA/genética , Desoxirribonucleotídeos/metabolismo , Humanos , Cinética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...