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1.
J Vet Diagn Invest ; 31(4): 640-644, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31170890

RESUMO

We developed a loop-mediated isothermal amplification (LAMP)-fluorescent loop primer (FLP) assay for genotyping the A/G2254 single nucleotide polymorphism (SNP) in the viral DNA polymerase gene of species Equid alphaherpesvirus 1 (EHV-1), which is associated with the neuropathogenic potential of this virus. In addition to the use of regular LAMP primers to amplify the target region, a 5'-FAM-labeled backward loop primer (FLB) and 3'-dabcyl-labeled quencher probe (QP) were designed for annealing curve analysis of the amplification product. The QP, which contacts the FLB, is located at the SNP site and has the A2254 allele. LAMP reactions were performed at 63°C for 40 min, and the subsequent annealing curve analyses were accomplished within 20 min. The LAMP-FLP assay could clearly differentiate A2254 and G2254 genotypes according to the difference in the annealing temperature of the QP between the 2 genotypes. Good agreement between the LAMP-FLP and the real-time PCR for genotyping of this SNP was observed in the detection of EHV-1 in equine clinical samples. The newly developed assay is a simple and rapid method for detecting and differentiating EHV-1 strains with A2254 and G2254 polymorphisms and would be suitable for clinical use.


Assuntos
DNA Viral/genética , DNA Polimerase Dirigida por DNA/genética , Herpesvirus Equídeo 1/genética , Técnicas de Amplificação de Ácido Nucleico/veterinária , Polimorfismo de Nucleotídeo Único , Animais , Genótipo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade
2.
BMC Plant Biol ; 19(1): 241, 2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-31170927

RESUMO

BACKGROUND: Plant chloroplasts and mitochondria utilize nuclear encoded proteins to replicate their DNA. These proteins are purposely built for replication in the organelle environment and are distinct from those involved in replication of the nuclear genome. These organelle-localized proteins have ancestral roots in bacterial and bacteriophage genes, supporting the endosymbiotic theory of their origin. We examined the interactions between three of these proteins from Arabidopsis thaliana: a DNA helicase-primase similar to bacteriophage T7 gp4 protein and animal mitochondrial Twinkle, and two DNA polymerases, Pol1A and Pol1B. We used a three-pronged approach to analyze the interactions, including Yeast-two-hybrid analysis, Direct Coupling Analysis (DCA), and thermophoresis. RESULTS: Yeast-two-hybrid analysis reveals residues 120-295 of Twinkle as the minimal region that can still interact with Pol1A or Pol1B. This region is a part of the primase domain of the protein and slightly overlaps the zinc-finger and RNA polymerase subdomains located within. Additionally, we observed that Arabidopsis Twinkle interacts much more strongly with Pol1A versus Pol1B. Thermophoresis also confirms that the primase domain of Twinkle has higher binding affinity than any other region of the protein. Direct-Coupling-Analysis identified specific residues in Twinkle and the DNA polymerases critical to positive interaction between the two proteins. CONCLUSIONS: The interaction of Twinkle with Pol1A or Pol1B mimics the minimal DNA replisomes of T7 phage and those present in mammalian mitochondria. However, while T7 and mammals absolutely require their homolog of Twinkle DNA helicase-primase, Arabidopsis Twinkle mutants are seemingly unaffected by this loss. This implies that while Arabidopsis mitochondria mimic minimal replisomes from T7 and mammalian mitochondria, there is an extra level of redundancy specific to loss of Twinkle function.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Bacteriófago T7/genética , DNA Polimerase Dirigida por DNA/genética , Complexos Multienzimáticos/genética , Enzimas Multifuncionais/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Mitocôndrias/metabolismo , Enzimas Multifuncionais/metabolismo
3.
Biol Pharm Bull ; 42(5): 764-769, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31061318

RESUMO

Werner helicase-interacting protein 1 (WRNIP1) was originally identified as a protein that interacts with WRN, the product of the gene responsible for Werner syndrome. Our previous studies suggested that WRNIP1 is implicated in translesion synthesis (TLS), a process in which specialized TLS polymerases replace replicative DNA polymerase and take over DNA synthesis on damaged templates. We proposed that a novel error-free pathway involving DNA polymerase δ and primase-polymerase (PrimPol) functions to synthesize DNA on UV-damaged DNA templates in the absence of WRNIP1 and the TLS polymerase Polη. Hence, in the current study, we analyzed the relationship between WRNIP1 and PrimPol. We found that WRNIP1 and PrimPol form a complex in cells. PrimPol protein expression was reduced in cells overexpressing WRNIP1, but was increased in WRNIP1-depleted cells. The WRNIP1-mediated reduction in the amount of PrimPol was suppressed by treatment of the cells with proteasome inhibitors, suggesting that WRNIP1 is involved in the degradation of PrimPol via the proteasome.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , DNA Primase/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Enzimas Multifuncionais/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , DNA Primase/genética , DNA Complementar/genética , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , Células HEK293 , Humanos , Enzimas Multifuncionais/genética , Plasmídeos , Inibidores de Proteassoma/farmacologia , RNA Mensageiro/metabolismo , Transfecção
4.
BMC Infect Dis ; 19(1): 388, 2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068147

RESUMO

BACKGROUND: The compound letermovir (LMV) has recently been approved for the prophylaxis of cytomegalovirus (CMV) infection and disease in adult CMV seropositive recipients of an allogeneic hematopoietic stem cell transplant. LMV inhibits CMV replication by binding to the viral terminase complex. However, first cases of clinical LMV resistance have been occurred. Here we report a fast breakthrough of resistant cytomegalovirus during secondary LMV prophylaxis in a hematopoietic-cell transplant recipient. CASE PRESENTATION: A 44-year-old male patient with acute myeloid leukemia (AML) experienced a CMV-reactivation within the first 4 weeks of allogeneic hematopoietic-cell transplantation. Administration of LMV was initiated at day + 34. Due to increasing viral loads, LMV treatment was discontinued after 8 days. The patient was then administered with valganciclovir (valGCV) until viral DNA was undetectable. Due to neutropenia, valGCV treatment was switched to LMV secondary prophylaxis. For 4 weeks, the patient maintain virologic suppression. Then, CMV viral loads increased with a fast kinetic. Genotypic testing of the viral polymerase UL54, the kinase UL97 as well as the viral terminase UL56 and UL89 revealed the mutation C325Y in UL56, which is associated with the high level LMV resistance. CONCLUSION: It is known that Letermovir is approved for prophylactic purposes. However, it may be used for some patients with CMV infection who either have failed prior therapies or are unable to tolerate other anti-CMV compounds. Particularly, the administration of LMV should be avoided in patients with detectable viral loads. When this is not possible, viral load must be routinely monitored along with UL56 genotyping. Furthermore, LMV administration at high virus loads may foster the rapid selection of resistant CMV mutants.


Assuntos
Acetatos/uso terapêutico , Antivirais/uso terapêutico , Infecções por Citomegalovirus/prevenção & controle , Citomegalovirus/efeitos dos fármacos , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Quinazolinas/uso terapêutico , Adulto , Citomegalovirus/genética , Infecções por Citomegalovirus/tratamento farmacológico , Infecções por Citomegalovirus/virologia , DNA Polimerase Dirigida por DNA/genética , Farmacorresistência Viral/efeitos dos fármacos , Humanos , Masculino , Mutação , Prevenção Secundária , Valganciclovir/uso terapêutico , Carga Viral/efeitos dos fármacos , Proteínas Virais/genética , Proteínas Estruturais Virais/genética
5.
Curr Microbiol ; 76(7): 863-871, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31069463

RESUMO

Although bacteriophages are the most abundant biological entities on the planet, their genetic diversity, especially in natural wetlands, is poorly understood. In this study, the genetic diversity of cyanopodoviruses in sediments of two coastal wetlands in Northeast China was investigated by targeting the DNA polymerase (pol) gene. A total of 66 DNA pol clones were obtained. A BLAST search at the amino acid level showed that the obtained sequences had the highest identity ranged from 83 to 99% to the known sequences. A phylogenetic tree showed that the distribution patterns of DNA pol sequence were different between two wetland soils, and 29 clones of this study formed four wetland-specific groups, which suggested that unrevealed novel groups of cyanopodovirus inhabited in wetlands. In addition, nonmetric multidimensional scaling (NMDS) analysis of all DNA pol sequences from various environments showed that cyanopodovirus communities of coastal wetlands are in the intermediate position between marine water environments and terrestrial freshwater environments, which highlights that the coastal wetlands as transitional zones between inland freshwater environments and marine environments.


Assuntos
Cianobactérias/virologia , Variação Genética , Podoviridae/classificação , Podoviridae/genética , Áreas Alagadas , China , DNA Viral/genética , DNA Polimerase Dirigida por DNA/genética , Estuários , Sedimentos Geológicos/virologia , Filogenia , Análise de Sequência de DNA , Microbiologia do Solo , Proteínas Virais/genética
6.
Talanta ; 201: 419-425, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31122444

RESUMO

Detection and identification of DNA by PCR has opened tremendous possibilities and allows detection of minute quantities of DNA highly specifically. However, PCR remains confined to laboratory settings because of the need of thermocyclers and other analytical equipment. This led to development of isothermal amplification techniques, among which Pad Lock Probe (PLP)-based Rolling Circle Amplification (RCA) has several advantages, but typically also requires a laboratory apparatus of some sort to measure DNA amplification. To circumvent this limitation, while still taking advantage of PLP-based RCA, we developed a colorimetric assay that relies on pH change. Using this assay, we can detect DNA in the low picomolar range and obtain results observable with the naked eye in only 20 min without any requirement for a thermocycler or other complex device, making it a particularly portable assay.


Assuntos
Técnicas Biossensoriais/métodos , Colorimetria/métodos , DNA Viral/sangue , Técnicas de Amplificação de Ácido Nucleico/métodos , Animais , Bacteriófago M13/genética , Calibragem , DNA Viral/genética , DNA Polimerase Dirigida por DNA/genética , Concentração de Íons de Hidrogênio , Indicadores e Reagentes/química , Virus da Influenza A Subtipo H5N1/genética , Limite de Detecção , Hibridização de Ácido Nucleico , Fenolsulfonaftaleína/química , Polimorfismo de Nucleotídeo Único , Ratos
7.
Virus Res ; 266: 52-57, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30991090

RESUMO

DNA polymerase (DNApol) is highly conserved in baculovirus and is required for viral DNA replication. However, little is known about gammabaculovirus DNApol. Here DNApol of the gammabaculovirus Neodiprion lecontei nucleopolyhedrovirus (NeleNPV) was cloned into a dnapol-null alphabaculovirus AcMNPV bacmid, creating Bac-GFP-AcΔPol-NlPol. The resulting recombinant bacmid did not spread to neighboring cells, virus growth curve and real-time PCR revealed that NeleNPV dnapol substitution did not rescue AcMNPV DNA replication and virus production. Immunofluorescence microscopy revealed that NeleNPV DNApol was expressed but could not localize to the nucleus. Subsequently NeleNPV DNApol was fused to SpltNPV DNApol nuclear localization signal (NLS) and the fused DNApol could import into nucleus. The NLS-fusing NeleNPV DNApol was further transposed into the dnapol-null AcMNPV bacmid, creating Bac-GFP-AcΔPol-HA:NlPolNLS. The recombinant virus could replicate and produce infectious virus in Sf9 cells, albeit at reduced levels compared to wild type AcMNPV. Taken together, our results suggested that the NLS deficiency of NeleNPV DNApol blocked viral DNA replication and production of infectious virus in dnapol-null AcMNPV bacmid.


Assuntos
DNA Viral/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Sinais de Localização Nuclear/deficiência , Nucleopolyhedrovirus/enzimologia , Proteínas Virais/metabolismo , Animais , Núcleo Celular/metabolismo , Replicação do DNA , DNA Viral/genética , DNA Polimerase Dirigida por DNA/deficiência , DNA Polimerase Dirigida por DNA/genética , Sinais de Localização Nuclear/genética , Nucleopolyhedrovirus/genética , Nucleopolyhedrovirus/fisiologia , Células Sf9 , Proteínas Virais/genética , Replicação Viral
8.
Nat Chem ; 11(6): 533-542, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31011171

RESUMO

The physicochemical properties of nucleic acids are dominated by their highly charged phosphodiester backbone chemistry. This polyelectrolyte structure decouples information content (base sequence) from bulk properties, such as solubility, and has been proposed as a defining trait of all informational polymers. However, this conjecture has not been tested experimentally. Here, we describe the encoded synthesis of a genetic polymer with an uncharged backbone chemistry: alkyl phosphonate nucleic acids (phNAs) in which the canonical, negatively charged phosphodiester is replaced by an uncharged P-alkyl phosphonodiester backbone. Using synthetic chemistry and polymerase engineering, we describe the enzymatic, DNA-templated synthesis of P-methyl and P-ethyl phNAs, and the directed evolution of specific streptavidin-binding phNA aptamer ligands directly from random-sequence mixed P-methyl/P-ethyl phNA repertoires. Our results establish an example of the DNA-templated enzymatic synthesis and evolution of an uncharged genetic polymer and provide a foundational methodology for their exploration as a source of novel functional molecules.


Assuntos
DNA/química , Organofosfonatos/química , Aptâmeros de Nucleotídeos/química , DNA/síntese química , DNA/genética , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , Evolução Molecular Direcionada/métodos , Mutação , Conformação de Ácido Nucleico , Organofosfonatos/síntese química , Engenharia de Proteínas/métodos , Estreptavidina/química , Thermococcaceae/enzimologia , Thermococcales/enzimologia
9.
Mol Cell ; 74(4): 785-800.e7, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-30948267

RESUMO

Antibiotics can induce mutations that cause antibiotic resistance. Yet, despite their importance, mechanisms of antibiotic-promoted mutagenesis remain elusive. We report that the fluoroquinolone antibiotic ciprofloxacin (cipro) induces mutations by triggering transient differentiation of a mutant-generating cell subpopulation, using reactive oxygen species (ROS). Cipro-induced DNA breaks activate the Escherichia coli SOS DNA-damage response and error-prone DNA polymerases in all cells. However, mutagenesis is limited to a cell subpopulation in which electron transfer together with SOS induce ROS, which activate the sigma-S (σS) general-stress response, which allows mutagenic DNA-break repair. When sorted, this small σS-response-"on" subpopulation produces most antibiotic cross-resistant mutants. A U.S. Food and Drug Administration (FDA)-approved drug prevents σS induction, specifically inhibiting antibiotic-promoted mutagenesis. Further, SOS-inhibited cell division, which causes multi-chromosome cells, promotes mutagenesis. The data support a model in which within-cell chromosome cooperation together with development of a "gambler" cell subpopulation promote resistance evolution without risking most cells.


Assuntos
Antibacterianos/efeitos adversos , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Mutagênese/genética , Divisão Celular/efeitos dos fármacos , Ciprofloxacino/efeitos adversos , Dano ao DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/genética , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/patogenicidade , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Mutagênese/efeitos dos fármacos , Mutação , Espécies Reativas de Oxigênio/metabolismo , Resposta SOS (Genética)/efeitos dos fármacos , Fator sigma/genética
10.
Cancer Biomark ; 24(3): 363-370, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30829610

RESUMO

BACKGROUND AND OBJECTIVE: Dysregulation of DNA polymerase iota (Pol ι) in breast cancer might contribute to the accumulation of genomic mutations and promotes breast cancer progression. In this study we explored the clinical relevance and biological function of Pol ι in breast cancer. METHODS: qRT-PCR was used to determine the expression levels of Pol ι in 31 breast cancer tissues. Then the stable overexpression of Pol ι and knockdown of Pol ι breast cancer cell lines were constructed. Wound-healing assay and transwell assay were performed to evaluate cell migratory and invasiveness, respectively. Signaling pathway was analyzed by western blot. RESULTS: The expression levels of Pol ι is overexpressed in breast cancer tissues and significantly higher in breast cancer tissues with lymph node metastasis compared to those without lymph node metastasis. Elevated Pol ι expression promoted migratory and invasiveness of breast cancer cells. Signaling pathway analysis indicated EGFR-ERK cascade works as a mediator of Pol ι-induced EMT of breast cancer cells. CONCLUSIONS: These data demonstrate the underlying mechanism by which Pol ι promotes breast cancer progression, suggesting that Pol ι may be a potential therapeutic target against breast cancer.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , DNA Polimerase Dirigida por DNA/metabolismo , Transição Epitelial-Mesenquimal , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Adulto , Idoso , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular , DNA Polimerase Dirigida por DNA/genética , Transição Epitelial-Mesenquimal/genética , Receptores ErbB/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Metástase Linfática , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Transdução de Sinais
11.
J Biol Chem ; 294(15): 6073-6081, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30842261

RESUMO

Classical DNA and RNA polymerase (pol) enzymes have defined roles with their respective substrates, but several pols have been found to have multiple functions. We reported previously that purified human DNA pol η (hpol η) can incorporate both deoxyribonucleoside triphosphates (dNTPs) and ribonucleoside triphosphates (rNTPs) and can use both DNA and RNA as substrates. X-ray crystal structures revealed that two pol η residues, Phe-18 and Tyr-92, behave as steric gates to influence sugar selectivity. However, the physiological relevance of these phenomena has not been established. Here, we show that purified hpol η adds rNTPs to DNA primers at physiological rNTP concentrations and in the presence of competing dNTPs. When two rATPs were inserted opposite a cyclobutane pyrimidine dimer, the substrate was less efficiently cleaved by human RNase H2. Human XP-V fibroblast extracts, devoid of hpol η, could not add rNTPs to a DNA primer, but the expression of transfected hpol η in the cells restored this ability. XP-V cell extracts did not add dNTPs to DNA primers hybridized to RNA, but could when hpol η was expressed in the cells. HEK293T cell extracts could add dNTPs to DNA primers hybridized to RNA, but lost this ability if hpol η was deleted. Interestingly, a similar phenomenon was not observed when other translesion synthesis (TLS) DNA polymerases-hpol ι, κ, or ζ-were individually deleted. These results suggest that hpol η is one of the major reverse transcriptases involved in physiological processes in human cells.


Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA , DNA Polimerase Dirigida por RNA , Transcrição Reversa , Linhagem Celular , Cristalografia por Raios X , Primers do DNA/química , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo
12.
DNA Repair (Amst) ; 76: 40-49, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30818168

RESUMO

DNA polymerases influence genome stability through their involvement in DNA replication, response to DNA damage, and DNA repair processes. Saccharomyces cerevisiae possess four non-essential DNA polymerases, Pol λ, Pol η, Pol ζ, and Rev1, which have varying roles in genome stability. In order to assess the contribution of the non-essential DNA polymerases in genome stability, we analyzed the pol4Δ rev1Δ rev3Δ rad30Δ quadruple mutant in microhomology mediated repair, due to recent studies linking some of these DNA polymerases to this repair pathway. Our results suggest that the length and quality of microhomology influence both the overall efficiency of repair and the involvement of DNA polymerases. Furthermore, the non-essential DNA polymerases demonstrate overlapping and redundant functions when repairing double-strand breaks using short microhomologies containing mismatches. Then, we examined genome-wide mutation accumulation in the pol4Δ rev1Δ rev3Δ rad30Δ quadruple mutant compared to wild type cells. We found a significant decrease in the overall rate of mutation accumulation in the quadruple mutant cells compared to wildtype, but an increase in frameshift mutations and a shift towards transversion base-substitution with a preference for G:C to T:A or C:G. Thus, the non-essential DNA polymerases have an impact on the nature of the mutational spectrum. The sequence and functional homology shared between human and S. cerevisiae non-essential DNA polymerases suggest these DNA polymerases may have a similar role in human cells.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Instabilidade Genômica , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Reparo do DNA , DNA Polimerase Dirigida por DNA/genética , Mutação
13.
Zhonghua Zhong Liu Za Zhi ; 41(2): 112-117, 2019 Feb 23.
Artigo em Chinês | MEDLINE | ID: mdl-30862140

RESUMO

Objective: To investigate the associations between genetic variations of DNA polymerase kappa (POLK) and treatment response to platinum-based chemotherapy of small cell lung cancer (SCLC), and to analyze the influencing factors on survival. Methods: Five haplotype-tagging single nucleotide polymorphisms (htSNPs) of POLK were genotyped by Sequenom MassARRAY methods in 1 030 SCLC patients who received platinum-based chemotherapy, and had different response and survival time. The associations between SNPs and treatment response were analyzed by computing the odds ratios (ORs) and 95% confidence intervals (CIs) from logistic regression model. Cox regression was used for survival analysis between SNPs and overall survival by computing the hazard ratios (HRs) and 95% CIs. Results: Among 1 030 cases, 558 (54.2%) cases received cis-platinum and etoposide treatment while others treated with carboplatin and etoposide. Seven hundred and eighty eight patients were chemotherapy responders in the study with a response rate of 76.5%. The median follow-up time of these patients was 22.0 months. Patients were followed up to get their survival information. The median survival time of these patients was 22.5 months. Six hundred and seventy three patients (65.3%) had died by the last date of follow-up to get their survival information (Dec 21, 2017). Five htSNPs of POLK were not associated with the chemotherapy response of SCLC patients who received platinum-based chemotherapy (all P>0.05). Multivariate Cox proportional hazards regression model analysis showed that, rs73120833 of POLK was significantly associated with the overall survival (OS) of SCLC patients, compared with POLK rs73120833 T allele, C allele can prolong OS (adjusted HR=0.87, 95% CI=0.77-0.97, P=0.021). The remaining 4 SNPS, including rs10077427, rs3756558, rs4549504 and rs5744545, were not significantly associated with overall survival. Age≤56, KPS> 80, limited-stage, chemotherapy response and radiation therapy can remarkably prolong OS (all P<0.05). Conclusion: These results suggest that POLK genetic polymorphism rs73120833 plays an important role on the prognosis of SCLC patients, which can be potential genetic biomarker for SCLC personalized treatment.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , DNA Polimerase Dirigida por DNA/genética , Variação Genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/genética , Cisplatino/administração & dosagem , Etoposídeo/administração & dosagem , Humanos , Neoplasias Pulmonares/mortalidade , Platina , Polimorfismo de Nucleotídeo Único , Prognóstico , Análise de Regressão , Carcinoma de Pequenas Células do Pulmão/mortalidade , Resultado do Tratamento
14.
Mol Cell ; 73(5): 915-929.e6, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30849395

RESUMO

DNA replication errors generate complex chromosomal rearrangements and thereby contribute to tumorigenesis and other human diseases. One mechanism that triggers these errors is mitotic entry before the completion of DNA replication. To address how mitosis might affect DNA replication, we used Xenopus egg extracts. When mitotic CDK (Cyclin B1-CDK1) is used to drive interphase egg extracts into a mitotic state, the replicative CMG (CDC45/MCM2-7/GINS) helicase undergoes ubiquitylation on its MCM7 subunit, dependent on the E3 ubiquitin ligase TRAIP. Whether replisomes have stalled or undergone termination, CMG ubiquitylation is followed by its extraction from chromatin by the CDC48/p97 ATPase. TRAIP-dependent CMG unloading during mitosis is also seen in C. elegans early embryos. At stalled forks, CMG removal results in fork breakage and end joining events involving deletions and templated insertions. Our results identify a mitotic pathway of global replisome disassembly that can trigger replication fork collapse and DNA rearrangements.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclina B1/metabolismo , Dano ao DNA , Replicação do DNA , DNA/biossíntese , Rearranjo Gênico , Mitose , Proteínas Quinases/metabolismo , Proteínas de Xenopus/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/genética , Ciclina B1/genética , DNA/genética , Reparo do DNA , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Manutenção de Minicromossomo/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/metabolismo
15.
Genes Dev ; 33(5-6): 282-287, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30808656

RESUMO

Here we show that translesion synthesis (TLS) opposite 1,N6-ethenodeoxyadenosine (εdA), which disrupts Watson-Crick base pairing, occurs via Polι/Polζ-, Rev1-, and Polθ-dependent pathways. The requirement of Polι/Polζ is consistent with the ability of Polι to incorporate nucleotide opposite εdA by Hoogsteen base pairing and of Polζ to extend synthesis. Rev1 polymerase and Polθ conduct TLS opposite εdA via alternative error-prone pathways. Strikingly, in contrast to extremely error-prone TLS opposite εdA by purified Polθ, it performs predominantly error-free TLS in human cells. Reconfiguration of the active site opposite εdA would provide Polθ the proficiency for error-free TLS in human cells.


Assuntos
Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Desoxiadenosinas/metabolismo , Domínio Catalítico , Adutos de DNA/metabolismo , Humanos
16.
Oncogene ; 38(22): 4310-4324, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30705406

RESUMO

Translesion DNA synthesis (TLS) and homologous recombination (HR) cooperate during S-phase to safeguard replication forks integrity. Thus, the inhibition of TLS becomes a promising point of therapeutic intervention in HR-deficient cancers, where TLS impairment might trigger synthetic lethality (SL). The main limitation to test this hypothesis is the current lack of selective pharmacological inhibitors of TLS. Herein, we developed a miniaturized screening assay to identify inhibitors of PCNA ubiquitylation, a key post-translational modification required for efficient TLS activation. After screening a library of 627 kinase inhibitors, we found that targeting the pro-survival kinase AKT leads to strong impairment of PCNA ubiquitylation. Mechanistically, we found that AKT-mediated modulation of Proliferating Cell Nuclear Antigen (PCNA) ubiquitylation after UV requires the upstream activity of DNA PKcs, without affecting PCNA ubiquitylation levels in unperturbed cells. Moreover, we confirmed that persistent AKT inhibition blocks the recruitment of TLS polymerases to sites of DNA damage and impairs DNA replication forks processivity after UV irradiation, leading to increased DNA replication stress and cell death. Remarkably, when we compared the differential survival of HR-proficient vs HR-deficient cells, we found that the combination of UV irradiation and AKT inhibition leads to robust SL induction in HR-deficient cells. We link this phenotype to AKT ability to inhibit PCNA ubiquitylation, since the targeted knockdown of PCNA E3-ligase (RAD18) and a non-ubiquitylable (PCNA K164R) knock-in model recapitulate the observed SL induction. Collectively, this work identifies AKT as a novel regulator of PCNA ubiquitylation and provides the proof-of-concept of inhibiting TLS as a therapeutic approach to selectively kill HR-deficient cells submitted to replication stress.


Assuntos
Replicação do DNA/genética , Recombinação Homóloga/genética , Antígeno Nuclear de Célula em Proliferação/genética , Proteínas Proto-Oncogênicas c-akt/genética , Ubiquitinação/genética , Morte Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , DNA/genética , Dano ao DNA/genética , DNA Polimerase Dirigida por DNA/genética , Células HCT116 , Células HEK293 , Humanos , Ubiquitina-Proteína Ligases/genética
17.
PLoS Genet ; 15(2): e1007956, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30716079

RESUMO

Mutagenic translesion DNA polymerase V (UmuD'2C) is induced as part of the DNA damage-induced SOS response in Escherichia coli, and is subjected to multiple levels of regulation. The UmuC subunit is sequestered on the cell membrane (spatial regulation) and enters the cytosol after forming a UmuD'2C complex, ~ 45 min post-SOS induction (temporal regulation). However, DNA binding and synthesis cannot occur until pol V interacts with a RecA nucleoprotein filament (RecA*) and ATP to form a mutasome complex, pol V Mut = UmuD'2C-RecA-ATP. The location of RecA relative to UmuC determines whether pol V Mut is catalytically on or off (conformational regulation). Here, we present three interrelated experiments to address the biochemical basis of conformational regulation. We first investigate dynamic deactivation during DNA synthesis and static deactivation in the absence of DNA synthesis. Single-molecule (sm) TIRF-FRET microscopy is then used to explore multiple aspects of pol V Mut dynamics. Binding of ATP/ATPγS triggers a conformational switch that reorients RecA relative to UmuC to activate pol V Mut. This process is required for polymerase-DNA binding and synthesis. Both dynamic and static deactivation processes are governed by temperature and time, in which on → off switching is "rapid" at 37°C (~ 1 to 1.5 h), "slow" at 30°C (~ 3 to 4 h) and does not require ATP hydrolysis. Pol V Mut retains RecA in activated and deactivated states, but binding to primer-template (p/t) DNA occurs only when activated. Studies are performed with two forms of the polymerase, pol V Mut-RecA wt, and the constitutively induced and hypermutagenic pol V Mut-RecA E38K/ΔC17. We discuss conformational regulation of pol V Mut, determined from biochemical analysis in vitro, in relation to the properties of pol V Mut in RecA wild-type and SOS constitutive genetic backgrounds in vivo.


Assuntos
DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Recombinases Rec A/metabolismo , Trifosfato de Adenosina/metabolismo , Dano ao DNA , DNA Bacteriano/biossíntese , DNA Polimerase Dirigida por DNA/genética , Ativação Enzimática , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Transferência Ressonante de Energia de Fluorescência , Genes Bacterianos , Cinética , Mutação , Conformação Proteica , Resposta SOS (Genética)
18.
Mol Cell ; 73(5): 900-914.e9, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30733119

RESUMO

Post-replication repair (PRR) allows tolerance of chemical- and UV-induced DNA base lesions in both an error-free and an error-prone manner. In classical PRR, PCNA monoubiquitination recruits translesion synthesis (TLS) DNA polymerases that can replicate through lesions. We find that PRR responds to DNA replication stress that does not cause base lesions. Rad5 forms nuclear foci during normal S phase and after exposure to types of replication stress where DNA base lesions are likely absent. Rad5 binds to the sites of stressed DNA replication forks, where it recruits TLS polymerases to repair single-stranded DNA (ssDNA) gaps, preventing mitotic defects and chromosome breaks. In contrast to the prevailing view of PRR, our data indicate that Rad5 promotes both mutagenic and error-free repair of undamaged ssDNA that arises during physiological and exogenous replication stress.


Assuntos
Quebras de DNA de Cadeia Simples , DNA Helicases/metabolismo , Reparo do DNA , Replicação do DNA , DNA Fúngico/metabolismo , DNA de Cadeia Simples/metabolismo , Mutação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Sítios de Ligação , Cromossomos Fúngicos , DNA Helicases/genética , DNA Fúngico/genética , DNA de Cadeia Simples/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Mitose , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Reparo de DNA por Recombinação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitinação
19.
J Vet Med Sci ; 81(3): 504-507, 2019 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-30700652

RESUMO

Elephant endotheliotropic herpesvirus type 1 (EEHV1) is the most important causative agent of an acute fatal hemorrhagic disease in Asian elephants (Elephas maximus). We employed loop-mediated isothermal amplification (LAMP) to develop a rapid and simple detection method for EEHV1 in blood. When used to test 21 clinical samples collected in Japan, the EEHV1 assay correctly identified one positive and 20 negative clinical samples. It was observed that when samples were spiked with synthetic DNA plasmids including EEHV1 polymerase gene, the detection limit of the LAMP assay was 101.2 copies/µl and 100-fold higher than that of conventional PCR. These advantages of the LAMP assay for EEHV1 detection may facilitate better veterinary practices for treating elephants suffering from the acute disease.


Assuntos
Elefantes/virologia , Infecções por Herpesviridae/veterinária , Herpesviridae/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/veterinária , Animais , Primers do DNA , DNA Polimerase Dirigida por DNA/genética , Elefantes/sangue , Feminino , Herpesviridae/classificação , Herpesviridae/enzimologia , Herpesviridae/genética , Infecções por Herpesviridae/diagnóstico , Infecções por Herpesviridae/virologia , Masculino , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/veterinária , Sensibilidade e Especificidade , Fatores de Tempo
20.
Nucleic Acids Res ; 47(6): 2966-2980, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30657944

RESUMO

Chain-terminating nucleoside analogs (CTNAs), which cannot be extended by DNA polymerases, are widely used as antivirals or anti-cancer agents, and can induce cell death. Processing of blocked DNA ends, like camptothecin-induced trapped-topoisomerase I, can be mediated by TDP1, BRCA1, CtIP and MRE11. Here, we investigated whether the CtIP-BRCA1 complex and MRE11 also contribute to cellular tolerance to CTNAs, including 2',3'-dideoxycytidine (ddC), cytarabine (ara-C) and zidovudine (Azidothymidine, AZT). We show that BRCA1-/-, CtIPS332A/-/- and nuclease-dead MRE11D20A/- mutants display increased sensitivity to CTNAs, accumulate more DNA damage (chromosomal breaks, γ-H2AX and neutral comets) when treated with CTNAs and exhibit significant delays in replication fork progression during exposure to CTNAs. Moreover, BRCA1-/-, CtIPS332A/-/- and nuclease-dead MRE11D20A/- mutants failed to resume DNA replication in response to CTNAs, whereas control and CtIP+/-/- cells experienced extensive recovery of DNA replication. In summary, we provide clear evidence that MRE11 and the collaborative action of BRCA1 and CtIP play a critical role in the nuclease-dependent removal of incorporated ddC from replicating genomic DNA. We propose that BRCA1-CTIP and MRE11 prepare nascent DNA ends, blocked from synthesis by CTNAs, for further repair.


Assuntos
Proteína BRCA1/genética , Proteínas de Transporte/genética , Proteína Homóloga a MRE11/genética , Proteínas Nucleares/genética , Terminação Traducional da Cadeia Peptídica , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/genética , Humanos , Nucleosídeos/análogos & derivados , Nucleosídeos/genética
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