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1.
Nucleic Acids Res ; 50(2): 962-974, 2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-35037018

RESUMO

We report the properties of two mutations in the exonuclease domain of the Saccharomyces cerevisiae DNA polymerase ϵ. One, pol2-Y473F, increases the mutation rate by about 20-fold, similar to the catalytically dead pol2-D290A/E290A mutant. The other, pol2-N378K, is a stronger mutator. Both retain the ability to excise a nucleotide from double-stranded DNA, but with impaired activity. pol2-Y473F degrades DNA poorly, while pol2-N378K degrades single-stranded DNA at an elevated rate relative to double-stranded DNA. These data suggest that pol2-Y473F reduces the capacity of the enzyme to perform catalysis in the exonuclease active site, while pol2-N378K impairs partitioning to the exonuclease active site. Relative to wild-type Pol ϵ, both variants decrease the dNTP concentration required to elicit a switch between proofreading and polymerization by more than an order of magnitude. While neither mutation appears to alter the sequence specificity of polymerization, the N378K mutation stimulates polymerase activity, increasing the probability of incorporation and extension of a mismatch. Considered together, these data indicate that impairing the primer strand transfer pathway required for proofreading increases the probability of common mutations by Pol ϵ, elucidating the association of homologous mutations in human DNA polymerase ϵ with cancer.


Assuntos
DNA Polimerase II/metabolismo , DNA Fúngico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Replicação do DNA , Mutação , Taxa de Mutação
2.
Proc Natl Acad Sci U S A ; 116(8): 3062-3071, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30718408

RESUMO

Mutations accumulate within somatic cells and have been proposed to contribute to aging. It is unclear what level of mutation burden may be required to consistently reduce cellular lifespan. Human cancers driven by a mutator phenotype represent an intriguing model to test this hypothesis, since they carry the highest mutation burdens of any human cell. However, it remains technically challenging to measure the replicative lifespan of individual mammalian cells. Here, we modeled the consequences of cancer-related mutator phenotypes on lifespan using yeast defective for mismatch repair (MMR) and/or leading strand (Polε) or lagging strand (Polδ) DNA polymerase proofreading. Only haploid mutator cells with significant lifetime mutation accumulation (MA) exhibited shorter lifespans. Diploid strains, derived by mating haploids of various genotypes, carried variable numbers of fixed mutations and a range of mutator phenotypes. Some diploid strains with fewer than two mutations per megabase displayed a 25% decrease in lifespan, suggesting that moderate numbers of random heterozygous mutations can increase mortality rate. As mutation rates and burdens climbed, lifespan steadily eroded. Strong diploid mutator phenotypes produced a form of genetic anticipation with regard to aging, where the longer a lineage persisted, the shorter lived cells became. Using MA lines, we established a relationship between mutation burden and lifespan, as well as population doubling time. Our observations define a threshold of random mutation burden that consistently decreases cellular longevity in diploid yeast cells. Many human cancers carry comparable mutation burdens, suggesting that while cancers appear immortal, individual cancer cells may suffer diminished lifespan due to accrued mutation burden.


Assuntos
Envelhecimento/genética , Reparo do DNA/genética , Longevidade/genética , Neoplasias/genética , Envelhecimento/patologia , Reparo de Erro de Pareamento de DNA/genética , Replicação do DNA/genética , Genótipo , Humanos , Mutação/genética , Acúmulo de Mutações , Taxa de Mutação , Neoplasias/patologia , Fenótipo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Sequenciamento Completo do Genoma
3.
PLoS Genet ; 11(4): e1005151, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25868109

RESUMO

Mutator phenotypes accelerate the evolutionary process of neoplastic transformation. Historically, the measurement of mutation rates has relied on scoring the occurrence of rare mutations in target genes in large populations of cells. Averaging mutation rates over large cell populations assumes that new mutations arise at a constant rate during each cell division. If the mutation rate is not constant, an expanding mutator population may contain subclones with widely divergent rates of evolution. Here, we report mutation rate measurements of individual cell divisions of mutator yeast deficient in DNA polymerase ε proofreading and base-base mismatch repair. Our data are best fit by a model in which cells can assume one of two distinct mutator states, with mutation rates that differ by an order of magnitude. In error-prone cell divisions, mutations occurred on the same chromosome more frequently than expected by chance, often in DNA with similar predicted replication timing, consistent with a spatiotemporal dimension to the hypermutator state. Mapping of mutations onto predicted replicons revealed that mutations were enriched in the first half of the replicon as well as near termination zones. Taken together, our findings show that individual genome replication events exhibit an unexpected volatility that may deepen our understanding of the evolution of mutator-driven malignancies.


Assuntos
Taxa de Mutação , Leveduras/genética , Alelos , Pareamento Incorreto de Bases , Divisão Celular , Reparo de Erro de Pareamento de DNA , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Replicação do DNA , DNA Fúngico/genética , Genoma Fúngico , Fenótipo , Replicon , Análise de Sequência de DNA , Leveduras/metabolismo
4.
Proc Natl Acad Sci U S A ; 112(19): E2457-66, 2015 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-25827226

RESUMO

Mutator phenotypes create genetic diversity that fuels tumor evolution. DNA polymerase (Pol) ε mediates leading strand DNA replication. Proofreading defects in this enzyme drive a number of human malignancies. Here, using budding yeast, we show that mutator variants of Pol ε depend on damage uninducible (Dun)1, an S-phase checkpoint kinase that maintains dNTP levels during a normal cell cycle and up-regulates dNTP synthesis upon checkpoint activation. Deletion of DUN1 (dun1Δ) suppresses the mutator phenotype of pol2-4 (encoding Pol ε proofreading deficiency) and is synthetically lethal with pol2-M644G (encoding altered Pol ε base selectivity). Although pol2-4 cells cycle normally, pol2-M644G cells progress slowly through S-phase. The pol2-M644G cells tolerate deletions of mediator of the replication checkpoint (MRC) 1 (mrc1Δ) and radiation sensitive (Rad) 9 (rad9Δ), which encode mediators of checkpoint responses to replication stress and DNA damage, respectively. The pol2-M644G mutator phenotype is partially suppressed by mrc1Δ but not rad9Δ; neither deletion suppresses the pol2-4 mutator phenotype. Thus, checkpoint activation augments the Dun1 effect on replication fidelity but is not required for it. Deletions of genes encoding key Dun1 targets that negatively regulate dNTP synthesis, suppress the dun1Δ pol2-M644G synthetic lethality and restore the mutator phenotype of pol2-4 in dun1Δ cells. DUN1 pol2-M644G cells have constitutively high dNTP levels, consistent with checkpoint activation. In contrast, pol2-4 and POL2 cells have similar dNTP levels, which decline in the absence of Dun1 and rise in the absence of the negative regulators of dNTP synthesis. Thus, dNTP pool levels correlate with Pol ε mutator severity, suggesting that treatments targeting dNTP pools could modulate mutator phenotypes for therapy.


Assuntos
DNA Polimerase Dirigida por DNA/genética , Mutação , Nucleotídeos/química , Fosfatos/química , Saccharomyces cerevisiae/metabolismo , Alelos , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Ciclo Celular , Análise Mutacional de DNA , Replicação do DNA , Variação Genética , Humanos , Mutagênese , Neoplasias/tratamento farmacológico , Neoplasias/genética , Fenótipo , Plasmídeos/metabolismo , Fase S , Saccharomyces cerevisiae/genética
5.
Hum Mutat ; 36(11): 1070-9, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26172944

RESUMO

Segmental progeroid syndromes are rare, heterogeneous disorders characterized by signs of premature aging affecting more than one tissue or organ. A prototypic example is the Werner syndrome (WS), caused by biallelic germline mutations in the Werner helicase gene (WRN). While heterozygous lamin A/C (LMNA) mutations are found in a few nonclassical cases of WS, another 10%-15% of patients initially diagnosed with WS do not have mutations in WRN or LMNA. Germline POLD1 mutations were recently reported in five patients with another segmental progeroid disorder: mandibular hypoplasia, deafness, progeroid features syndrome. Here, we describe eight additional patients with heterozygous POLD1 mutations, thereby substantially expanding the characterization of this new example of segmental progeroid disorders. First, we identified POLD1 mutations in patients initially diagnosed with WS. Second, we describe POLD1 mutation carriers without clinically relevant hearing impairment or mandibular underdevelopment, both previously thought to represent obligate diagnostic features. These patients also exhibit a lower incidence of metabolic abnormalities and joint contractures. Third, we document postnatal short stature and premature greying/loss of hair in POLD1 mutation carriers. We conclude that POLD1 germline mutations can result in a variably expressed and probably underdiagnosed segmental progeroid syndrome.


Assuntos
Síndrome de Cockayne/diagnóstico , Síndrome de Cockayne/genética , DNA Polimerase III/genética , Mutação em Linhagem Germinativa , Síndrome de Werner/diagnóstico , Adolescente , Adulto , Alelos , Substituição de Aminoácidos , Linhagem Celular Transformada , Criança , Instabilidade Cromossômica , Aberrações Cromossômicas , Análise Mutacional de DNA , DNA Polimerase III/química , Diagnóstico Diferencial , Fácies , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Fenótipo , Conformação Proteica , Sistema de Registros , Adulto Jovem
6.
PLoS Genet ; 7(10): e1002282, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22022273

RESUMO

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.


Assuntos
DNA Polimerase III/metabolismo , Replicação do DNA/genética , Instabilidade de Microssatélites , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Supressão Genética/genética , Alelos , Animais , Dano ao DNA/genética , DNA Polimerase III/genética , Reparo do DNA/genética , Escherichia coli/genética , Genótipo , Haploidia , Camundongos , Taxa de Mutação , Proteínas de Saccharomyces cerevisiae/genética
7.
Crit Rev Biochem Mol Biol ; 46(6): 548-70, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21977975

RESUMO

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.


Assuntos
DNA Polimerase Dirigida por DNA/genética , Mutação , Animais , Reparo de Erro de Pareamento de DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/química , Variação Genética , Humanos , Modelos Moleculares , Conformação Proteica
8.
Semin Cancer Biol ; 20(5): 281-93, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20951805

RESUMO

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.


Assuntos
Aberrações Cromossômicas , Reparo do DNA/genética , Replicação do DNA/genética , Mutação , Neoplasias/genética , Animais , Dano ao DNA , Reparo de Erro de Pareamento de DNA , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Rearranjo Gênico , Humanos , Instabilidade de Microssatélites , Mutação Puntual
9.
Elife ; 102021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34523420

RESUMO

Although studies of Saccharomyces cerevisiae have provided many insights into mutagenesis and DNA repair, most of this work has focused on a few laboratory strains. Much less is known about the phenotypic effects of natural variation within S. cerevisiae's DNA repair pathways. Here, we use natural polymorphisms to detect historical mutation spectrum differences among several wild and domesticated S. cerevisiae strains. To determine whether these differences are likely caused by genetic mutation rate modifiers, we use a modified fluctuation assay with a CAN1 reporter to measure de novo mutation rates and spectra in 16 of the analyzed strains. We measure a 10-fold range of mutation rates and identify two strains with distinctive mutation spectra. These strains, known as AEQ and AAR, come from the panel's 'Mosaic beer' clade and share an enrichment for C > A mutations that is also observed in rare variation segregating throughout the genomes of several Mosaic beer and Mixed origin strains. Both AEQ and AAR are haploid derivatives of the diploid natural isolate CBS 1782, whose rare polymorphisms are enriched for C > A as well, suggesting that the underlying mutator allele is likely active in nature. We use a plasmid complementation test to show that AAR and AEQ share a mutator allele in the DNA repair gene OGG1, which excises 8-oxoguanine lesions that can cause C > A mutations if left unrepaired.


Assuntos
Variação Genética , Mutação Puntual , Saccharomyces cerevisiae/genética , Alelos , Sistemas de Transporte de Aminoácidos Básicos/genética , Reparo do DNA , Diploide , Teste de Complementação Genética , Haploidia , Taxa de Mutação , Fenótipo , Saccharomyces cerevisiae/classificação , Proteínas de Saccharomyces cerevisiae/genética
10.
Commun Biol ; 4(1): 21, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33398111

RESUMO

Mutations that compromise mismatch repair (MMR) or DNA polymerase ε or δ exonuclease domains produce mutator phenotypes capable of fueling cancer evolution. Here, we investigate how combined defects in these pathways expands genetic heterogeneity in cells of the budding yeast, Saccharomyces cerevisiae, using a single-cell resolution approach that tallies all mutations arising from individual divisions. The distribution of replication errors present in mother cells after the initial S-phase was broader than expected for a single uniform mutation rate across all cell divisions, consistent with volatility of the mutator phenotype. The number of mismatches that then segregated to the mother and daughter cells co-varied, suggesting that each division is governed by a different underlying genome-wide mutation rate. The distribution of mutations that individual cells inherit after the second S-phase is further broadened by the sequential actions of semiconservative replication and mitotic segregation of chromosomes. Modeling suggests that this asymmetric segregation may diversify mutation burden in mutator-driven tumors.


Assuntos
Taxa de Mutação , Alelos , Reparo de Erro de Pareamento de DNA/genética , DNA Polimerase II/genética , Heterogeneidade Genética , Saccharomyces cerevisiae , Software
11.
Genetics ; 215(4): 959-974, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32513814

RESUMO

Mutations affecting DNA polymerase exonuclease domains or mismatch repair (MMR) generate "mutator" phenotypes capable of driving tumorigenesis. Cancers with both defects exhibit an explosive increase in mutation burden that appears to reach a threshold, consistent with selection acting against further mutation accumulation. In Saccharomyces cerevisiae haploid yeast, simultaneous defects in polymerase proofreading and MMR select for "antimutator" mutants that suppress the mutator phenotype. We report here that spontaneous polyploids also escape this "error-induced extinction" and routinely outcompete antimutators in evolved haploid cultures. We performed similar experiments to explore how diploid yeast adapt to the mutator phenotype. We first evolved cells with homozygous mutations affecting polymerase δ proofreading and MMR, which we anticipated would favor tetraploid emergence. While tetraploids arose with a low frequency, in most cultures, a single antimutator clone rose to prominence carrying biallelic mutations affecting the polymerase mutator alleles. Variation in mutation rate between subclones from the same culture suggests that there exists continued selection pressure for additional antimutator alleles. We then evolved diploid yeast modeling MMR-deficient cancers with the most common heterozygous exonuclease domain mutation (POLE-P286R). Although these cells grew robustly, within 120 generations, all subclones carried truncating or nonsynonymous mutations in the POLE-P286R homologous allele (pol2-P301R) that suppressed the mutator phenotype as much as 100-fold. Independent adaptive events in the same culture were common. Our findings suggest that analogous tumor cell populations may adapt to the threat of extinction by polyclonal mutations that neutralize the POLE mutator allele and preserve intratumoral genetic diversity for future adaptation.


Assuntos
Adaptação Fisiológica , Evolução Molecular , Genoma Fúngico , Poliploidia , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Mutação , Taxa de Mutação , Fenótipo , Saccharomyces cerevisiae/crescimento & desenvolvimento
12.
Geroscience ; 42(2): 749-764, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31975050

RESUMO

The loss of vacuolar/lysosomal acidity is an early event during aging that has been linked to mitochondrial dysfunction. However, it is unclear how loss of vacuolar acidity results in age-related dysfunction. Through unbiased genetic screens, we determined that increased iron uptake can suppress the mitochondrial respiratory deficiency phenotype of yeast vma mutants, which have lost vacuolar acidity due to genetic disruption of the vacuolar ATPase proton pump. Yeast vma mutants exhibited nuclear localization of Aft1, which turns on the iron regulon in response to iron-sulfur cluster (ISC) deficiency. This led us to find that loss of vacuolar acidity with age in wild-type yeast causes ISC defects and a DNA damage response. Using microfluidics to investigate aging at the single-cell level, we observe grossly divergent trajectories of iron homeostasis within an isogenic and environmentally homogeneous population. One subpopulation of cells fails to mount the expected compensatory iron regulon gene expression program, and suffers progressively severe ISC deficiency with little to no activation of the iron regulon. In contrast, other cells show robust iron regulon activity with limited ISC deficiency, which allows extended passage and survival through a period of genomic instability during aging. These divergent trajectories suggest that iron regulation and ISC homeostasis represent a possible target for aging interventions.


Assuntos
Homeostase , Ferro , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Ferro/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Enxofre
13.
Sci Rep ; 7: 46535, 2017 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-28417960

RESUMO

In yeast, the pol3-01,L612M double mutant allele, which causes defects in DNA polymerase delta (Pol δ) proofreading (pol3-01) and nucleotide selectivity (pol3-L612M), confers an "ultramutator" phenotype that rapidly drives extinction of haploid and diploid MMR-proficient cells. Here, we investigate antimutator mutations that encode amino acid substitutions in Pol δ that suppress this lethal phenotype. We find that most of the antimutator mutations individually suppress the pol3-01 and pol3-L612M mutator phenotypes. The locations of many of the amino acid substitutions in Pol δ resemble those of previously identified antimutator substitutions; however, two novel mutations encode substitutions (R674G and Q697R) of amino acids in the fingers domain that coordinate the incoming dNTP. These mutations are lethal without pol3-L612M and markedly change the mutation spectra produced by the pol3-01,L612M mutator allele, suggesting that they alter nucleotide selection to offset the pol3-L612M mutator phenotype. Consistent with this hypothesis, mutations and drug treatments that perturb dNTP pool levels disproportionately influence the viability of pol3-L612M,R674G and pol3-L612M,Q697R cells. Taken together, our findings suggest that mutation rate can evolve through genetic changes that alter the balance of dNTP binding and dissociation from DNA polymerases.


Assuntos
Substituição de Aminoácidos , DNA Polimerase III/metabolismo , Mutação de Sentido Incorreto , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , DNA Polimerase III/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
14.
FEBS Lett ; 579(26): 5879-88, 2005 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-16162339
15.
Genetics ; 196(3): 677-91, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24388879

RESUMO

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.


Assuntos
DNA Polimerase III/genética , Replicação do DNA , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimologia , Reparo de Erro de Pareamento de DNA , DNA Polimerase III/metabolismo , Diploide , Genes Essenciais , Genes Fúngicos , Genoma Fúngico , Taxa de Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise de Sequência de DNA
16.
Genetics ; 193(3): 751-70, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23307893

RESUMO

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.


Assuntos
DNA Polimerase II/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Reparo de Erro de Pareamento de DNA/genética , DNA Polimerase II/química , DNA Polimerase II/metabolismo , Replicação do DNA/genética , Dados de Sequência Molecular , Taxa de Mutação , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
17.
Mech Ageing Dev ; 133(4): 118-26, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22079405

RESUMO

The somatic mutation theory of aging posits that the accumulation of mutations in the genetic material of somatic cells as a function of time results in a decrease in cellular function. In particular, the accumulation of random mutations may inactivate genes that are important for the functioning of the somatic cells of various organ systems of the adult, result in a decrease in organ function. When the organ function decreases below a critical level, death occurs. A significant amount of research has shown that somatic mutations play an important role in aging and a number of age related pathologies. In this review, we explore evidence for increases in somatic nuclear mutation burden with age and the consequences for aging, cancer, and neurodegeneration. We then review evidence for increases in mitochondrial mutation burden and the consequences for dysfunction in the disease processes.


Assuntos
Envelhecimento/genética , DNA Mitocondrial , Mutação , Neoplasias/genética , Doenças Neurodegenerativas/genética , Fatores Etários , Envelhecimento/patologia , Animais , Predisposição Genética para Doença , Humanos , Neoplasias/patologia , Doenças Neurodegenerativas/patologia , Fenótipo
18.
Plant Cell ; 19(5): 1507-21, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17526749

RESUMO

The silencing phenotype in Arabidopsis thaliana lines with an inverted repeat transgene under the control of a phloem-specific promoter was manifested in regions around veins due to a mobile signal of silencing. Genetic analysis implicates RNA-DEPENDENT RNA POLYMERASE2 (RDR2) and an RNA polymerase IVa subunit gene (NRPD1a) in the signaling mechanism. We also identified an SNF2 domain-containing protein (CLASSY1) that acts together with RDR2 and NRPD1a in the spread of transgene silencing and in the production of endogenous 24-nucleotide short interfering RNAs (siRNAs). Cytochemical analysis indicates that CLASSY1 may act in the nucleus with NRPD1a and RDR2 in the upstream part of RNA silencing pathways that generate a double-stranded RNA substrate for Dicer-like (DCL) nucleases. DCL3 and ARGONAUTE4 act in a downstream part of the pathway, leading to endogenous 24-nucleotide siRNA production, but are not required for intercellular signaling. From genetic analysis, we conclude that another downstream part of the pathway associated with intercellular signaling requires DCL4 and at least one other protein required for 21-nucleotide trans-acting siRNAs. We interpret the effect of polymerase IVa and trans-acting siRNA pathway mutations in terms of a modular property of RNA silencing pathways.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Interferência de RNA , Transdução de Sinais , Alelos , Sequência de Aminoácidos , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , DNA Polimerase beta/metabolismo , Genes Reporter , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , MicroRNAs/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese , Mutação/genética , Fenótipo , Plantas Geneticamente Modificadas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , RNA Interferente Pequeno/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Frações Subcelulares/metabolismo
19.
Proc Natl Acad Sci U S A ; 103(41): 14994-5001, 2006 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-17008405

RESUMO

Many eukaryotic cells use RNA-directed silencing mechanisms to protect against viruses and transposons and to suppress endogenous gene expression at the posttranscriptional level. RNA silencing also is implicated in epigenetic mechanisms affecting chromosome structure and transcriptional gene silencing. Here, we describe enhanced silencing phenotype (esp) mutants in Arabidopsis thaliana that reveal how proteins associated with RNA processing and 3' end formation can influence RNA silencing. These proteins were a putative DEAH RNA helicase homologue of the yeast PRP2 RNA splicing cofactor and homologues of mRNA 3' end formation proteins CstF64, symplekin/PTA1, and CPSF100. The last two proteins physically associated with the flowering time regulator FY in the 3' end formation complex AtCPSF. The phenotypes of the 3' end formation esp mutants include impaired termination of the transgene transcripts, early flowering, and enhanced silencing of the FCA-beta mRNA. Based on these findings, we propose that the ESP-containing 3' end formation complexes prevent transgene and endogenous mRNAs from entering RNA-silencing pathways. According to this proposal, in the absence of these ESP proteins, these RNAs have aberrant 3' termini. The aberrant RNAs would enter the RNA silencing pathways because they are converted into dsRNA by RNA-dependent RNA polymerases.


Assuntos
Arabidopsis/fisiologia , Flores/genética , Interferência de RNA , Processamento Pós-Transcricional do RNA/genética , RNA de Plantas/antagonistas & inibidores , RNA de Plantas/genética , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Fator de Especificidade de Clivagem e Poliadenilação/antagonistas & inibidores , Fator de Especificidade de Clivagem e Poliadenilação/genética , Fator Estimulador de Clivagem/antagonistas & inibidores , Fator Estimulador de Clivagem/genética , Endopeptidases/genética , Endopeptidases/metabolismo , Flores/fisiologia , Dados de Sequência Molecular , Mutação , RNA de Plantas/metabolismo , Separase , Homologia de Sequência de Aminoácidos
20.
J Biol Chem ; 279(12): 11081-7, 2004 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-14707145

RESUMO

This study investigates bypassing initiated from codons immediately 5' of a stop codon. The mRNA slips and is scanned by the peptidyl-tRNA for a suitable landing site, and standard decoding resumes at the next 3' codon. This work shows that landing sites with potentially strong base pairing between the peptidyl-tRNA anticodon and mRNA are preferred, but sites with little or no potential for Watson-Crick or wobble base pairing can also be utilized. These results have implications for re-pairing in ribosomal frameshifting. Shine-Dalgarno sequences in the mRNA can alter the distribution of landing sites observed. The bacteriophage T4 gene 60 nascent peptide, known to influence take-off in its native context, imposes stringent P-site pairing requirements, thereby limiting the number of suitable landing sites.


Assuntos
Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Sequência de Bases , Mudança da Fase de Leitura do Gene Ribossômico , Espectrometria de Massas , RNA Mensageiro/química , Aminoacil-RNA de Transferência/química
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