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1.
Proc Natl Acad Sci U S A ; 116(49): 24779-24785, 2019 12 03.
Artículo en Inglés | MEDLINE | ID: mdl-31748270

RESUMEN

The super-enhancers (SEs) of lineage-specific genes in B cells are off-target sites of somatic hypermutation. However, the inability to detect sufficient numbers of mutations in normal human B cells has precluded the generation of a high-resolution mutational landscape of SEs. Here we captured and sequenced 12 B cell SEs at single-nucleotide resolution from 10 healthy individuals across diverse ethnicities. We detected a total of approximately 9,000 subclonal mutations (allele frequencies <0.1%); of these, approximately 8,000 are present in the BCL6 SE alone. Within the BCL6 SE, we identified 3 regions of clustered mutations in which the mutation frequency is ∼7 × 10-4 Mutational spectra show a predominance of C > T/G > A and A > G/T > C substitutions, consistent with the activities of activation-induced-cytidine deaminase (AID) and the A-T mutator, DNA polymerase η, respectively, in mutagenesis in normal B cells. Analyses of mutational signatures further corroborate the participation of these factors in this process. Single base substitution signatures SBS85, SBS37, and SBS39 were found in the BCL6 SE. While SBS85 is a denoted signature of AID in lymphoid cells, the etiologies of SBS37 and SBS39 are unknown. Our analysis suggests the contribution of error-prone DNA polymerases to the latter signatures. The high-resolution mutation landscape has enabled accurate profiling of subclonal mutations in B cell SEs in normal individuals. By virtue of the fact that subclonal SE mutations are clonally expanded in B cell lymphomas, our studies also offer the potential for early detection of neoplastic alterations.


Asunto(s)
Linfocitos B/metabolismo , Elementos de Facilitación Genéticos/genética , Proteínas Proto-Oncogénicas c-bcl-6/genética , Adulto , Línea Celular , Citidina Desaminasa/genética , Análisis Mutacional de ADN/métodos , ADN Polimerasa Dirigida por ADN/genética , Frecuencia de los Genes , Sitios Genéticos/genética , Voluntarios Sanos , Humanos , Linfoma de Células B/sangre , Linfoma de Células B/diagnóstico , Linfoma de Células B/genética , Persona de Mediana Edad , Tasa de Mutación , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Adulto Joven
2.
Nat Methods ; 9(1): 78-80, 2011 Nov 27.
Artículo en Inglés | MEDLINE | ID: mdl-22120468

RESUMEN

Because mutations are inevitable, the genome of each cell in a multicellular organism becomes unique and therefore encodes a record of its ancestry. Here we coupled arbitrary single primer PCR with next-generation DNA sequencing to catalog mutations and deconvolve the phylogeny of cultured mouse cells. This study helps pave the way toward construction of retrospective cell-fate maps based on mutations accumulating in genomes of somatic cells.


Asunto(s)
Linaje de la Célula/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutación , Análisis de Secuencia de ADN/métodos , Animales , Simulación por Computador , Genoma , Ratones , Filogenia , Reacción en Cadena de la Polimerasa/métodos , Reproducibilidad de los Resultados
3.
PLoS Genet ; 7(10): e1002282, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22022273

RESUMEN

Cells rely on a network of conserved pathways to govern DNA replication fidelity. Loss of polymerase proofreading or mismatch repair elevates spontaneous mutation and facilitates cellular adaptation. However, double mutants are inviable, suggesting that extreme mutation rates exceed an error threshold. Here we combine alleles that affect DNA polymerase δ (Pol δ) proofreading and mismatch repair to define the maximal error rate in haploid yeast and to characterize genetic suppressors of mutator phenotypes. We show that populations tolerate mutation rates 1,000-fold above wild-type levels but collapse when the rate exceeds 10⁻³ inactivating mutations per gene per cell division. Variants that escape this error-induced extinction (eex) rapidly emerge from mutator clones. One-third of the escape mutants result from second-site changes in Pol δ that suppress the proofreading-deficient phenotype, while two-thirds are extragenic. The structural locations of the Pol δ changes suggest multiple antimutator mechanisms. Our studies reveal the transient nature of eukaryotic mutators and show that mutator phenotypes are readily suppressed by genetic adaptation. This has implications for the role of mutator phenotypes in cancer.


Asunto(s)
ADN Polimerasa III/metabolismo , Replicación del ADN/genética , Inestabilidad de Microsatélites , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Supresión Genética/genética , Alelos , Animales , Daño del ADN/genética , ADN Polimerasa III/genética , Reparación del ADN/genética , Escherichia coli/genética , Genotipo , Haploidia , Ratones , Tasa de Mutación , Proteínas de Saccharomyces cerevisiae/genética
4.
Crit Rev Biochem Mol Biol ; 46(6): 548-70, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21977975

RESUMEN

Evolution balances DNA replication speed and accuracy to optimize replicative fitness and genetic stability. There is no selective pressure to improve DNA replication fidelity beyond the background mutation rate from other sources, such as DNA damage. However, DNA polymerases remain amenable to amino acid substitutions that lower intrinsic error rates. Here, we review these 'antimutagenic' changes in DNA polymerases and discuss what they reveal about mechanisms of replication fidelity. Pioneering studies with bacteriophage T4 DNA polymerase (T4 Pol) established the paradigm that antimutator amino acid substitutions reduce replication errors by increasing proofreading efficiency at the expense of polymerase processivity. The discoveries of antimutator substitutions in proofreading-deficient 'mutator' derivatives of bacterial Pols I and III and yeast Pol δ suggest there must be additional antimutagenic mechanisms. Remarkably, many of the affected amino acid positions from Pol I, Pol III, and Pol δ are similar to the original T4 Pol substitutions. The locations of antimutator substitutions within DNA polymerase structures suggest that they may increase nucleotide selectivity and/or promote dissociation of primer termini from polymerases poised for misincorporation, leading to expulsion of incorrect nucleotides. If misincorporation occurs, enhanced primer dissociation from polymerase domains may improve proofreading in cis by an intrinsic exonuclease or in trans by alternate cellular proofreading activities. Together, these studies reveal that natural selection can readily restore replication error rates to sustainable levels following an adaptive mutator phenotype.


Asunto(s)
ADN Polimerasa Dirigida por ADN/genética , Mutación , Animales , Reparación de la Incompatibilidad de ADN , Replicación del ADN , ADN Polimerasa Dirigida por ADN/química , Variación Genética , Humanos , Modelos Moleculares , Conformación Proteica
5.
Nucleic Acids Res ; 39(11): e73, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21459851

RESUMEN

Herein, a detailed protocol for a random mutation capture (RMC) assay to measure nuclear point mutation frequency in mouse tissue is described. This protocol is a simplified version of the original method developed for human tissue that is easier to perform, yet retains a high sensitivity of detection. In contrast to assays relying on phenotypic selection of reporter genes in transgenic mice, the RMC assay allows direct detection of mutations in endogenous genes in any mouse strain. Measuring mutation frequency within an intron of a transcribed gene, we show this assay to be highly reproducible. We analyzed mutation frequencies from the liver tissue of animals with a mutation within the intrinsic exonuclease domains of the two major DNA polymerases, δ and ε. These mice exhibited significantly higher mutation frequencies than did wild-type animals. A comparison with a previous analysis of these genotypes in Big Blue mice revealed the RMC assay to be more sensitive than the Big Blue assay for this application. As RMC does not require analysis of a particular gene, simultaneous analysis of mutation frequency at multiple genetic loci is feasible. This assay provides a versatile alternative to transgenic mouse models for the study of mutagenesis in vivo.


Asunto(s)
Análisis Mutacional de ADN , Mutación Puntual , Animales , ADN Polimerasa II/genética , ADN Polimerasa III/genética , Genoma , Ratones , Ratones Endogámicos C57BL , Mutagénesis , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados
6.
Proc Natl Acad Sci U S A ; 106(40): 17101-4, 2009 Oct 06.
Artículo en Inglés | MEDLINE | ID: mdl-19805137

RESUMEN

Organisms require faithful DNA replication to avoid deleterious mutations. In yeast, replicative leading- and lagging-strand DNA polymerases (Pols epsilon and delta, respectively) have intrinsic proofreading exonucleases that cooperate with each other and mismatch repair to limit spontaneous mutation to less than 1 per genome per cell division. The relationship of these pathways in mammals and their functions in vivo are unknown. Here we show that mouse Pol epsilon and delta proofreading suppress discrete mutator and cancer phenotypes. We found that inactivation of Pol epsilon proofreading elevates base-substitution mutations and accelerates a unique spectrum of spontaneous cancers; the types of tumors are entirely different from those triggered by loss of Pol delta proofreading. Intercrosses of Pol epsilon-, Pol delta-, and mismatch repair-mutant mice show that Pol epsilon and delta proofreading act in parallel pathways to prevent spontaneous mutation and cancer. These findings distinguish Pol epsilon and delta functions in vivo and reveal tissue-specific requirements for DNA replication fidelity.


Asunto(s)
ADN Polimerasa III/genética , ADN Polimerasa II/genética , Mutación , Neoplasias/patología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Secuencia de Bases , ADN Polimerasa II/metabolismo , ADN Polimerasa III/metabolismo , Femenino , Perfilación de la Expresión Génica , Frecuencia de los Genes , Genotipo , Estimación de Kaplan-Meier , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos , Datos de Secuencia Molecular , Homólogo 1 de la Proteína MutL , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
7.
Semin Cancer Biol ; 20(5): 281-93, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20951805

RESUMEN

Cancer is fueled by mutations and driven by adaptive selection. Normal cells avoid deleterious mutations by replicating their genomes with extraordinary accuracy. Here we review the pathways governing DNA replication fidelity and discuss evidence implicating replication errors (point mutation instability or PIN) in carcinogenesis.


Asunto(s)
Aberraciones Cromosómicas , Reparación del ADN/genética , Replicación del ADN/genética , Mutación , Neoplasias/genética , Animales , Daño del ADN , Reparación de la Incompatibilidad de ADN , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Reordenamiento Génico , Humanos , Inestabilidad de Microsatélites , Mutación Puntual
8.
Mol Cell Biol ; 27(21): 7669-82, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17785453

RESUMEN

Mammalian DNA polymerase delta (Pol delta) is believed to replicate a large portion of the genome and to synthesize DNA in DNA repair and genetic recombination pathways. The effects of mutation in the polymerase domain of this essential enzyme are unknown. Here, we generated mice harboring an L604G or L604K substitution in highly conserved motif A in the polymerase active site of Pol delta. Homozygous Pold1(L604G/L604G) and Pold1(L604K/L604K) mice died in utero. However, heterozygous animals were viable and displayed no overall increase in disease incidence, indicative of efficient compensation for the defective mutant polymerase. The life spans of wild-type and heterozygous Pold1(+/L604G) mice did not differ, while that of Pold1(+/L604K) mice was reduced by 18%. Cultured embryonic fibroblasts from the heterozygous strains exhibited comparable increases in both spontaneous mutation rate and chromosome aberrations. We observed no significant increase in cancer incidence; however, Pold1(+/L604K) mice bearing histologically diagnosed tumors died at a younger median age than wild-type mice. Our results indicate that heterozygous mutation at L604 in the polymerase active site of DNA polymerase delta reduces life span, increases genomic instability, and accelerates tumorigenesis in an allele-specific manner, novel findings that have implications for human cancer.


Asunto(s)
Transformación Celular Neoplásica/genética , ADN Polimerasa III/química , ADN Polimerasa III/metabolismo , Inestabilidad Genómica , Mutación/genética , Neoplasias/enzimología , Neoplasias/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Células Cultivadas , Cromosomas de los Mamíferos/metabolismo , Daño del ADN , ADN Polimerasa III/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/enzimología , Fibroblastos/citología , Fibroblastos/enzimología , Heterocigoto , Histonas/metabolismo , Longevidad , Pérdida de Heterocigocidad , Ratones , Datos de Secuencia Molecular , Neoplasias/patología , Fenotipo , Fosforilación , Estructura Secundaria de Proteína , Análisis de Supervivencia
9.
Toxicol Pathol ; 38(3): 476-85, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20190201

RESUMEN

Acute tumor lysis syndrome (ATLS) is characterized by severe metabolic abnormalities and organ dysfunction resulting from rapid destruction of neoplastic cells. Metabolic disturbances are thought to be the primary cause of clinical ATLS symptoms, which include renal dysfunction, seizures, and cardiac arrhythmias. The histopathologic lesions associated with organ dysfunction are largely unknown because of the low rate of mortality of ATLS in humans and the few cases of ATLS identified in laboratory animals. Here, we describe histologic, immunohistochemical, and electron microscopic analyses of thirty-one ATLS cases from a cohort of 499 mice that are prone to spontaneous lymphoblastic lymphoma owing to genetic defects in DNA replication fidelity. Seventy-three percent of our cohort died with lymphoblastic lymphoma, and 8% of affected mice died with diffuse microthromboemboli consistent with ATLS. Mice with ATLS had a high spontaneous mortality rate (>50%), a large tumor burden with disseminated disease, and evidence of leukemia. Blood vessels in the lung, kidney, and other organs were occluded by microthromboemboli composed of chromatin, cellular debris, fibrin, platelets, and entrapped erythrocytes and malignant cells. This case series suggests that ATLS can occur at high frequency in mice with disseminated lymphoblastic lymphoma and leads to a high rate of spontaneous death from microthromboemboli.


Asunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras/complicaciones , Síndrome de Lisis Tumoral/patología , Síndrome de Lisis Tumoral/veterinaria , Animales , Reparación de la Incompatibilidad de ADN/genética , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Microscopía Electrónica de Transmisión , Tromboembolia/etiología , Tromboembolia/patología , Síndrome de Lisis Tumoral/genética
10.
Curr Biol ; 16(6): R209-11, 2006 Mar 21.
Artículo en Inglés | MEDLINE | ID: mdl-16546074

RESUMEN

Proofreading is the primary guardian of DNA polymerase fidelity. New work has revealed that polymerases with intrinsic proofreading activity may cooperate with non-proofreading polymerases to ensure faithful DNA replication.


Asunto(s)
Replicación del ADN/fisiología , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/metabolismo , Animales , Exonucleasas/metabolismo , Modelos Genéticos , Mutación
11.
Nucleic Acids Res ; 35(4): 1054-63, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17264132

RESUMEN

Base excision repair (BER) is the major pathway for the repair of simple, non-bulky lesions in DNA that is initiated by a damage-specific DNA glycosylase. Several human DNA glycosylases exist that efficiently excise numerous types of lesions, although the close proximity of a single strand break (SSB) to a DNA adduct can have a profound effect on both BER and SSB repair. We recently reported that DNA lesions located as a second nucleotide 5'-upstream to a DNA SSB are resistant to DNA glycosylase activity and this study further examines the processing of these 'complex' lesions. We first demonstrated that the damaged base should be excised before SSB repair can occur, since it impaired processing of the SSB by the BER enzymes, DNA ligase IIIalpha and DNA polymerase beta. Using human whole cell extracts, we next isolated the major activity against DNA lesions located as a second nucleotide 5'-upstream to a DNA SSB and identified it as DNA polymerase delta (Pol delta). Using recombinant protein we confirmed that the 3'-5'-exonuclease activity of Pol delta can efficiently remove these DNA lesions. Furthermore, we demonstrated that mouse embryonic fibroblasts, deficient in the exonuclease activity of Pol delta are partially deficient in the repair of these 'complex' lesions, demonstrating the importance of Pol delta during the repair of DNA lesions in close proximity to a DNA SSB, typical of those induced by ionizing radiation.


Asunto(s)
Roturas del ADN de Cadena Simple , ADN Polimerasa III/metabolismo , Reparación del ADN , Animales , Células Cultivadas , ADN Ligasas/metabolismo , ADN Polimerasa III/genética , ADN Polimerasa beta/metabolismo , Exodesoxirribonucleasas/metabolismo , Células HeLa , Humanos , Hipoxantina/metabolismo , Ratones , Mutación , Uracilo/análogos & derivados , Uracilo/metabolismo
12.
Mol Cell Biol ; 25(14): 5823-33, 2005 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15988000

RESUMEN

Aflatoxin B1 (AFB1) is a human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In humans, AFB1 is primarily bioactivated by cytochrome P450 1A2 (CYP1A2) and 3A4 to a genotoxic epoxide that forms N7-guanine DNA adducts. A series of yeast haploid mutants defective in DNA repair and cell cycle checkpoints were transformed with human CYP1A2 to investigate how these DNA adducts are repaired. Cell survival and mutagenesis following aflatoxin B1 treatment was assayed in strains defective in nucleotide excision repair (NER) (rad14), postreplication repair (PRR) (rad6, rad18, mms2, and rad5), homologous recombinational repair (HRR) (rad51 and rad54), base excision repair (BER) (apn1 apn2), nonhomologous end-joining (NHEJ) (yku70), mismatch repair (MMR) (pms1), translesion synthesis (TLS) (rev3), and checkpoints (mec1-1, mec1-1 rad53, rad9, and rad17). Together our data suggest the involvement of homologous recombination and nucleotide excision repair, postreplication repair, and checkpoints in the repair and/or tolerance of AFB1-induced DNA damage in the yeast model. Rev3 appears to mediate AFB1-induced mutagenesis when error-free pathways are compromised. The results further suggest unique roles for Rad5 and abasic endonuclease-dependent DNA intermediates in regulating AFB1-induced mutagenicity.


Asunto(s)
Aflatoxina B1/toxicidad , Sistema Enzimático del Citocromo P-450/metabolismo , Daño del ADN , Reparación del ADN/fisiología , Saccharomyces cerevisiae/genética , Aflatoxina B1/metabolismo , Disparidad de Par Base/genética , Carcinógenos/metabolismo , Carcinógenos/toxicidad , Sistema Enzimático del Citocromo P-450/genética , Reparación del ADN/genética , Replicación del ADN/genética , Humanos , Mutación , Recombinación Genética/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología
13.
Virus Res ; 107(2): 215-28, 2005 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-15649567

RESUMEN

HIV-1 and other retroviruses exhibit mutation rates that are 1,000,000-fold greater than their host organisms. Error-prone viral replication may place retroviruses and other RNA viruses near the threshold of "error catastrophe" or extinction due to an intolerable load of deleterious mutations. Strategies designed to drive viruses to error catastrophe have been applied to HIV-1 and a number of RNA viruses. Here, we review the concept of extinguishing HIV infection by "lethal mutagenesis" and consider the utility of this new approach in combination with conventional antiretroviral strategies.


Asunto(s)
VIH-1/efectos de los fármacos , Mutágenos/farmacología , Mutación , Replicación Viral , Desaminasa APOBEC-3G , Antivirales/farmacología , Citidina Desaminasa , VIH-1/genética , VIH-1/fisiología , Humanos , Nucleósido Desaminasas , Nucleósidos/química , Nucleósidos/farmacología , Proteínas/metabolismo , Proteínas Represoras
14.
Curr Med Res Opin ; 31(3): 557-74, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25651481

RESUMEN

OBJECTIVE: Tumor necrosis factor (TNF) is a highly pleiotropic cytokine with multiple activities other than its originally discovered role of tumor necrosis in rodents. TNF is now understood to play a contextual role in driving either tumor elimination or promotion. Using both animal and human data, this review examines the role of TNF in cancer development and the effect of TNF and TNF inhibitors (TNFis) on malignancy risk. RESEARCH DESIGN: A literature review was performed using relevant search terms for TNF and malignancy. RESULTS: Although administration of TNF can cause tumor regression in specific rodent tumor models, human expression polymorphisms suggest that TNF can be a tumor-promoting cytokine, whereas blocking the TNF pathway in a variety of tumor models inhibits tumor growth. In addition to direct effects of TNF on tumors, TNF can variously affect immunity and the tumor microenvironment. Whereas TNF can promote immune surveillance designed to eliminate tumors, it can also drive chronic inflammation, autoimmunity, angiogenesis, and other processes that promote tumor initiation, growth, and spread. Key players in TNF signaling that shape this response include NF-κB and JNK, and malignant-inflammatory cell interactions, each of which may have different responses to TNF signaling. Focusing on rheumatoid arthritis (RA) patients, where clinical experience is most extensive, a review of the clinical literature shows no increased risk of overall malignancy or solid tumors such as breast and lung cancers with exposure to TNFis. Lymphoma rates are not increased with use of TNFis. Conflicting data exist regarding the risks of melanoma and nonmelanoma skin cancer. Data regarding the risk of recurrent malignancy are limited. CONCLUSIONS: Overall, the available data indicate that elevated TNF is a risk factor for cancer, whereas its inhibition in RA patients is not generally associated with an increased cancer risk. In particular, TNF inhibition is not associated with cancers linked to immune suppression. A better understanding of the tumor microenvironment, molecular events underlying specific tumors, and epidemiologic studies of malignancies within specific disease indications should enable more focused pharmacovigilance studies and a better understanding of the potential risks of TNFis.


Asunto(s)
Artritis Reumatoide/inmunología , Inflamación/inmunología , Neoplasias , Factores de Necrosis Tumoral , Animales , Modelos Animales de Enfermedad , Humanos , Recurrencia Local de Neoplasia/metabolismo , Neoplasias/inmunología , Neoplasias/metabolismo , Factores de Riesgo , Transducción de Señal , Microambiente Tumoral , Inhibidores del Factor de Necrosis Tumoral , Factores de Necrosis Tumoral/metabolismo
15.
Genetics ; 196(3): 677-91, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24388879

RESUMEN

Genetic defects in DNA polymerase accuracy, proofreading, or mismatch repair (MMR) induce mutator phenotypes that accelerate adaptation of microbes and tumor cells. Certain combinations of mutator alleles synergistically increase mutation rates to levels that drive extinction of haploid cells. The maximum tolerated mutation rate of diploid cells is unknown. Here, we define the threshold for replication error-induced extinction (EEX) of diploid Saccharomyces cerevisiae. Double-mutant pol3 alleles that carry mutations for defective DNA polymerase-δ proofreading (pol3-01) and accuracy (pol3-L612M or pol3-L612G) induce strong mutator phenotypes in heterozygous diploids (POL3/pol3-01,L612M or POL3/pol3-01,L612G). Both pol3-01,L612M and pol3-01,L612G alleles are lethal in the homozygous state; cells with pol3-01,L612M divide up to 10 times before arresting at random stages in the cell cycle. Antimutator eex mutations in the pol3 alleles suppress this lethality (pol3-01,L612M,eex or pol3-01,L612G,eex). MMR defects synergize with pol3-01,L612M,eex and pol3-01,L612G,eex alleles, increasing mutation rates and impairing growth. Conversely, inactivation of the Dun1 S-phase checkpoint kinase suppresses strong pol3-01,L612M,eex and pol3-01,L612G,eex mutator phenotypes as well as the lethal pol3-01,L612M phenotype. Our results reveal that the lethal error threshold in diploids is 10 times higher than in haploids and likely determined by homozygous inactivation of essential genes. Pronounced loss of fitness occurs at mutation rates well below the lethal threshold, suggesting that mutator-driven cancers may be susceptible to drugs that exacerbate replication errors.


Asunto(s)
ADN Polimerasa III/genética , Replicación del ADN , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimología , Reparación de la Incompatibilidad de ADN , ADN Polimerasa III/metabolismo , Diploidia , Genes Esenciales , Genes Fúngicos , Genoma Fúngico , Tasa de Mutación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/metabolismo , Análisis de Secuencia de ADN
16.
Genetics ; 193(3): 751-70, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23307893

RESUMEN

DNA polymerases (Pols) ε and δ perform the bulk of yeast leading- and lagging-strand DNA synthesis. Both Pols possess intrinsic proofreading exonucleases that edit errors during polymerization. Rare errors that elude proofreading are extended into duplex DNA and excised by the mismatch repair (MMR) system. Strains that lack Pol proofreading or MMR exhibit a 10- to 100-fold increase in spontaneous mutation rate (mutator phenotype), and inactivation of both Pol δ proofreading (pol3-01) and MMR is lethal due to replication error-induced extinction (EEX). It is unclear whether a similar synthetic lethal relationship exists between defects in Pol ε proofreading (pol2-4) and MMR. Using a plasmid-shuffling strategy in haploid Saccharomyces cerevisiae, we observed synthetic lethality of pol2-4 with alleles that completely abrogate MMR (msh2Δ, mlh1Δ, msh3Δ msh6Δ, or pms1Δ mlh3Δ) but not with partial MMR loss (msh3Δ, msh6Δ, pms1Δ, or mlh3Δ), indicating that high levels of unrepaired Pol ε errors drive extinction. However, variants that escape this error-induced extinction (eex mutants) frequently emerged. Five percent of pol2-4 msh2Δ eex mutants encoded second-site changes in Pol ε that reduced the pol2-4 mutator phenotype between 3- and 23-fold. The remaining eex alleles were extragenic to pol2-4. The locations of antimutator amino-acid changes in Pol ε and their effects on mutation spectra suggest multiple mechanisms of mutator suppression. Our data indicate that unrepaired leading- and lagging-strand polymerase errors drive extinction within a few cell divisions and suggest that there are polymerase-specific pathways of mutator suppression. The prevalence of suppressors extragenic to the Pol ε gene suggests that factors in addition to proofreading and MMR influence leading-strand DNA replication fidelity.


Asunto(s)
ADN Polimerasa II/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Reparación de la Incompatibilidad de ADN/genética , ADN Polimerasa II/química , ADN Polimerasa II/metabolismo , Replicación del ADN/genética , Datos de Secuencia Molecular , Tasa de Mutación , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
17.
DNA Repair (Amst) ; 9(1): 11-22, 2010 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-19896421

RESUMEN

Werner syndrome (WS) is a human autosomal recessive genetic instability and cancer predisposition syndrome with features of premature aging. Several genetically determined mouse models of WS have been generated, however, none develops features of premature aging or an elevated risk of neoplasia unless additional genetic perturbations are introduced. In order to determine whether differences in cellular phenotype could explain the discrepant phenotypes of Wrn-/- mice and WRN-deficient humans, we compared the cellular phenotype of newly derived Wrn-/- mouse primary fibroblasts with previous analyses of primary and transformed fibroblasts from WS patients and with newly derived, WRN-depleted human primary fibroblasts. These analyses confirmed previously reported cellular phenotypes of WRN-mutant and WRN-deficient human fibroblasts, and demonstrated that the human WRN-deficient cellular phenotype can be detected in cells grown in 5% or in 20% oxygen. In contrast, we did not identify prominent cellular phenotypes present in WRN-deficient human cells in Wrn-/- mouse fibroblasts. Our results indicate that human and mouse fibroblasts have different functional requirements for WRN protein, and that the absence of a strong cellular phenotype may in part explain the failure of Wrn-/- mice to develop an organismal phenotype resembling Werner syndrome.


Asunto(s)
Exodesoxirribonucleasas/metabolismo , RecQ Helicasas/metabolismo , Síndrome de Werner/enzimología , Animales , Proliferación Celular , Células Cultivadas , Daño del ADN , Exodesoxirribonucleasas/deficiencia , Histonas/metabolismo , Humanos , Longevidad , Ratones , Ratones Noqueados , Neoplasias/enzimología , Neoplasias/genética , Oxígeno/metabolismo , Fenotipo , RecQ Helicasas/deficiencia , Síndrome de Werner/genética , Síndrome de Werner/patología , Helicasa del Síndrome de Werner
18.
J Infect Dis ; 199(9): 1323-6, 2009 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-19358668

RESUMEN

Genotypic surveys suggest that human immunodeficiency virus type 1 (HIV-1) and HIV-2 evolve different sets of mutations in response to nucleoside reverse-transcriptase inhibitors (NRTIs). We used site-directed mutagenesis, culture-based phenotyping, and cell-free assays to determine the resistance profiles conferred by specific amino acid replacements in HIV-2 reverse transcriptase. Although thymidine analogue mutations had no effect on zidovudine sensitivity, the addition of Q151M together with K65R or M184V was sufficient for high-level resistance to both lamivudine and zidovudine in HIV-2, and the combination of K65R, Q151M, and M184V conferred classwide NRTI resistance. These data suggest that current NRTI-based regimens are suboptimal for treating HIV-2 infection.


Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Antivirales/farmacología , Infecciones por VIH/tratamiento farmacológico , VIH-2/genética , ADN Polimerasa Dirigida por ARN/genética , Inhibidores de la Transcriptasa Inversa/uso terapéutico , África Occidental/epidemiología , Sustitución de Aminoácidos , Fármacos Anti-VIH/farmacología , Farmacorresistencia Viral/efectos de los fármacos , Predisposición Genética a la Enfermedad , VIH-1/efectos de los fármacos , VIH-1/genética , VIH-2/efectos de los fármacos , VIH-2/enzimología , Humanos , Mutagénesis Sitio-Dirigida , Fenotipo , ADN Polimerasa Dirigida por ARN/efectos de los fármacos , Zidovudina/farmacología
19.
Antimicrob Agents Chemother ; 52(1): 329-32, 2008 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17967913

RESUMEN

Using an indicator cell assay that directly quantifies viral replication, we show that human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2, respectively) exhibit similar sensitivities to 3'-azido-3'-deoxythymidine (zidovudine) as well as other nucleoside analog inhibitors of reverse transcriptase. These data support the use of nucleoside analogs for antiviral therapy of HIV-2 infection.


Asunto(s)
Fármacos Anti-VIH/farmacología , VIH-1/efectos de los fármacos , VIH-2/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa/farmacología , Zidovudina/farmacología , VIH-1/fisiología , VIH-2/fisiología , Células HeLa , Humanos , Pruebas de Sensibilidad Microbiana/métodos
20.
J Virol ; 80(14): 7169-78, 2006 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-16809322

RESUMEN

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) contains four structural motifs (A, B, C, and D) that are conserved in polymerases from diverse organisms. Motif B interacts with the incoming nucleotide, the template strand, and key active-site residues from other motifs, suggesting that motif B is an important determinant of substrate specificity. To examine the functional role of this region, we performed "random scanning mutagenesis" of 11 motif B residues and screened replication-competent mutants for altered substrate analog sensitivity in culture. Single amino acid replacements throughout the targeted region conferred resistance to lamivudine and/or hypersusceptibility to zidovudine (AZT). Substitutions at residue Q151 increased the sensitivity of HIV-1 to multiple nucleoside analogs, and a subset of these Q151 variants was also hypersusceptible to the pyrophosphate analog phosphonoformic acid (PFA). Other AZT-hypersusceptible mutants were resistant to PFA and are therefore phenotypically similar to PFA-resistant variants selected in vitro and in infected patients. Collectively, these data show that specific amino acid replacements in motif B confer broad-spectrum hypersusceptibility to substrate analog inhibitors. Our results suggest that motif B influences RT-deoxynucleoside triphosphate interactions at multiple steps in the catalytic cycle of polymerization.


Asunto(s)
Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/genética , VIH-1/fisiología , Replicación Viral/genética , Secuencias de Aminoácidos/genética , Catálisis , Farmacorresistencia Viral/efectos de los fármacos , Farmacorresistencia Viral/genética , Foscarnet/análogos & derivados , Foscarnet/farmacología , Células HeLa , Humanos , Mutagénesis , Unión Proteica/genética , Estructura Terciaria de Proteína , Inhibidores de la Transcriptasa Inversa/farmacología , Especificidad por Sustrato/genética , Replicación Viral/efectos de los fármacos , Zidovudina/farmacología
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