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1.
Proc Natl Acad Sci U S A ; 116(35): 17377-17382, 2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31409704

RESUMO

Gross Chromosomal Rearrangements (GCRs) play an important role in human diseases, including cancer. Although most of the nonessential Genome Instability Suppressing (GIS) genes in Saccharomyces cerevisiae are known, the essential genes in which mutations can cause increased GCR rates are not well understood. Here 2 S. cerevisiae GCR assays were used to screen a targeted collection of temperature-sensitive mutants to identify mutations that caused increased GCR rates. This identified 94 essential GIS (eGIS) genes in which mutations cause increased GCR rates and 38 candidate eGIS genes that encode eGIS1 protein-interacting or family member proteins. Analysis of TCGA data using the human genes predicted to encode the proteins and protein complexes implicated by the S. cerevisiae eGIS genes revealed a significant enrichment of mutations affecting predicted human eGIS genes in 10 of the 16 cancers analyzed.

2.
Nat Commun ; 10(1): 3238, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324763

RESUMO

Leucine-rich repeat (LRR) domains are evolutionarily conserved in proteins that function in development and immunity. Here we report strict exonic modularity of LRR domains of several human gene families, which is a precondition for alternative splicing (AS). We provide evidence for AS of LRR domain within several Nod-like receptors, most prominently the inflammasome sensor NLRP3. Human NLRP3, but not mouse NLRP3, is expressed as two major isoforms, the full-length variant and a variant lacking exon 5. Moreover, NLRP3 AS is stochastically regulated, with NLRP3 ∆ exon 5 lacking the interaction surface for NEK7 and hence loss of activity. Our data thus reveals unexpected regulatory roles of AS through differential utilization of LRRs modules in vertebrate innate immunity.

4.
Cancer Cell ; 35(3): 504-518.e7, 2019 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-30827889

RESUMO

Ionizing radiation (IR) and chemotherapy are standard-of-care treatments for glioblastoma (GBM) patients and both result in DNA damage, however, the clinical efficacy is limited due to therapeutic resistance. We identified a mechanism of such resistance mediated by phosphorylation of PTEN on tyrosine 240 (pY240-PTEN) by FGFR2. pY240-PTEN is rapidly elevated and bound to chromatin through interaction with Ki-67 in response to IR treatment and facilitates the recruitment of RAD51 to promote DNA repair. Blocking Y240 phosphorylation confers radiation sensitivity to tumors and extends survival in GBM preclinical models. Y240F-Pten knockin mice showed radiation sensitivity. These results suggest that FGFR-mediated pY240-PTEN is a key mechanism of radiation resistance and is an actionable target for improving radiotherapy efficacy.

5.
J Biol Chem ; 293(47): 18055-18070, 2018 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-30237169

RESUMO

DNA mismatch repair (MMR) corrects mispaired DNA bases and small insertion/deletion loops generated by DNA replication errors. After binding a mispair, the eukaryotic mispair recognition complex Msh2-Msh6 binds ATP in both of its nucleotide-binding sites, which induces a conformational change resulting in the formation of an Msh2-Msh6 sliding clamp that releases from the mispair and slides freely along the DNA. However, the roles that Msh2-Msh6 sliding clamps play in MMR remain poorly understood. Here, using Saccharomyces cerevisiae, we created Msh2 and Msh6 Walker A nucleotide-binding site mutants that have defects in ATP binding in one or both nucleotide-binding sites of the Msh2-Msh6 heterodimer. We found that these mutations cause a complete MMR defect in vivo The mutant Msh2-Msh6 complexes exhibited normal mispair recognition and were proficient at recruiting the MMR endonuclease Mlh1-Pms1 to mispaired DNA. At physiological (2.5 mm) ATP concentration, the mutant complexes displayed modest partial defects in supporting MMR in reconstituted Mlh1-Pms1-independent and Mlh1-Pms1-dependent MMR reactions in vitro and in activation of the Mlh1-Pms1 endonuclease and showed a more severe defect at low (0.1 mm) ATP concentration. In contrast, five of the mutants were completely defective and one was mostly defective for sliding clamp formation at high and low ATP concentrations. These findings suggest that mispair-dependent sliding clamp formation triggers binding of additional Msh2-Msh6 complexes and that further recruitment of additional downstream MMR proteins is required for signal amplification of mispair binding during MMR.

6.
Nat Commun ; 9(1): 3680, 2018 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-30206225

RESUMO

Genome instability is associated with tumorigenesis. Here, we identify a role for the histone Htz1, which is deposited by the Swr1 chromatin-remodeling complex (SWR-C), in preventing genome instability in the absence of the replication fork/replication checkpoint proteins Mrc1, Csm3, or Tof1. When combined with deletion of SWR1 or HTZ1, deletion of MRC1, CSM3, or TOF1 or a replication-defective mrc1 mutation causes synergistic increases in gross chromosomal rearrangement (GCR) rates, accumulation of a broad spectrum of GCRs, and hypersensitivity to replication stress. The double mutants have severe replication defects and accumulate aberrant replication intermediates. None of the individual mutations cause large increases in GCR rates; however, defects in MRC1, CSM3 or TOF1 cause activation of the DNA damage checkpoint and replication defects. We propose a model in which Htz1 deposition and retention in chromatin prevents transiently stalled replication forks that occur in mrc1, tof1, or csm3 mutants from being converted to DNA double-strand breaks that trigger genome instability.

7.
Nat Struct Mol Biol ; 25(8): 650-659, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30061603

RESUMO

Eukaryotic DNA mismatch repair (MMR) involves both exonuclease 1 (Exo1)-dependent and Exo1-independent pathways. We found that the unstructured C-terminal domain of Saccharomyces cerevisiae Exo1 contains two MutS homolog 2 (Msh2)-interacting peptide (SHIP) boxes downstream from the MutL homolog 1 (Mlh1)-interacting peptide (MIP) box. These three sites were redundant in Exo1-dependent MMR in vivo and could be replaced by a fusion protein between an N-terminal fragment of Exo1 and Msh6. The SHIP-Msh2 interactions were eliminated by the msh2M470I mutation, and wild-type but not mutant SHIP peptides eliminated Exo1-dependent MMR in vitro. We identified two S. cerevisiae SHIP-box-containing proteins and three candidate human SHIP-box-containing proteins. One of these, Fun30, had a small role in Exo1-dependent MMR in vivo. The Remodeling of the Structure of Chromatin (Rsc) complex also functioned in both Exo1-dependent and Exo1-independent MMR in vivo. Our results identified two modes of Exo1 recruitment and a peptide module that mediates interactions between Msh2 and other proteins, and they support a model in which Exo1 functions in MMR by being tethered to the Msh2-Msh6 complex.

8.
PLoS Genet ; 14(3): e1007250, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29505562

RESUMO

Mms21, a subunit of the Smc5/6 complex, possesses an E3 ligase activity for the Small Ubiquitin-like MOdifier (SUMO). Here we show that the mms21-CH mutation, which inactivates Mms21 ligase activity, causes increased accumulation of gross chromosomal rearrangements (GCRs) selected in the dGCR assay. These dGCRs are formed by non-allelic homologous recombination between divergent DNA sequences mediated by Rad52-, Rrm3- and Pol32-dependent break-induced replication. Combining mms21-CH with sgs1Δ caused a synergistic increase in GCRs rates, indicating the distinct roles of Mms21 and Sgs1 in suppressing GCRs. The mms21-CH mutation also caused increased rates of accumulating uGCRs mediated by breakpoints in unique sequences as revealed by whole genome sequencing. Consistent with the accumulation of endogenous DNA lesions, mms21-CH mutants accumulate increased levels of spontaneous Rad52 and Ddc2 foci and had a hyper-activated DNA damage checkpoint. Together, these findings support that Mms21 prevents the accumulation of spontaneous DNA lesions that cause diverse GCRs.


Assuntos
Dano ao DNA/genética , Proteína SUMO-1/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Cromossomos Fúngicos , Reparo do DNA , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Epistasia Genética , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Genoma Fúngico , Mutação , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , RecQ Helicases/genética , RecQ Helicases/metabolismo , Proteína SUMO-1/genética , Proteínas de Saccharomyces cerevisiae/genética
9.
PLoS Genet ; 14(1): e1007170, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29320491

RESUMO

Defects in the genes encoding the Paf1 complex can cause increased genome instability. Loss of Paf1, Cdc73, and Ctr9, but not Rtf1 or Leo1, caused increased accumulation of gross chromosomal rearrangements (GCRs). Combining the cdc73Δ mutation with individual deletions of 43 other genes, including TEL1 and YKU80, which are involved in telomere maintenance, resulted in synergistic increases in GCR rates. Whole genome sequence analysis of GCRs indicated that there were reduced relative rates of GCRs mediated by de novo telomere additions and increased rates of translocations and inverted duplications in cdc73Δ single and double mutants. Analysis of telomere lengths and telomeric gene silencing in strains containing different combinations of cdc73Δ, tel1Δ and yku80Δ mutations suggested that combinations of these mutations caused increased defects in telomere maintenance. A deletion analysis of Cdc73 revealed that a central 105 amino acid region was necessary and sufficient for suppressing the defects observed in cdc73Δ strains; this region was required for the binding of Cdc73 to the Paf1 complex through Ctr9 and for nuclear localization of Cdc73. Taken together, these data suggest that the increased GCR rate of cdc73Δ single and double mutants is due to partial telomere dysfunction and that Ctr9 and Paf1 play a central role in the Paf1 complex potentially by scaffolding the Paf1 complex subunits or by mediating recruitment of the Paf1 complex to the different processes it functions in.


Assuntos
Instabilidade Genômica/genética , Proteínas Nucleares/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Homeostase do Telômero/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas Nucleares/genética , Organismos Geneticamente Modificados , Fenótipo , Ligação Proteica , Proteínas Serina-Treonina Quinases/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Telômero/metabolismo , Fatores de Elongação da Transcrição/genética
10.
Methods Mol Biol ; 1672: 43-61, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29043616

RESUMO

Genome rearrangements underlie different human diseases including many cancers. Determining the rates at which genome rearrangements arise and isolating unique, independent genome rearrangements is critical to understanding the genes and pathways that prevent or promote genome rearrangements. Here, we describe quantitative S. cerevisiae genetic assays for measuring the rates of accumulating genome rearrangements including deletions, translocations, and broken chromosomes healed by de novo telomere addition that result in the deletion of two counter-selectable genes, CAN1 and URA3, placed in the nonessential regions of the S. cerevisiae genome. The assays also allow for the isolation of individual genome rearrangements for structural studies, and a method for analyzing genome rearrangements by next-generation DNA sequencing is provided.


Assuntos
Genoma Fúngico , Genômica , Recombinação Genética , Saccharomyces cerevisiae/genética , Instabilidade Genômica , Biblioteca Genômica , Genômica/métodos , Deleção de Sequência , Translocação Genética , Sequenciamento Completo do Genoma
11.
Genetics ; 206(3): 1187-1225, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28684602

RESUMO

Genome rearrangements result in mutations that underlie many human diseases, and ongoing genome instability likely contributes to the development of many cancers. The tools for studying genome instability in mammalian cells are limited, whereas model organisms such as Saccharomyces cerevisiae are more amenable to these studies. Here, we discuss the many genetic assays developed to measure the rate of occurrence of Gross Chromosomal Rearrangements (called GCRs) in S. cerevisiae These genetic assays have been used to identify many types of GCRs, including translocations, interstitial deletions, and broken chromosomes healed by de novo telomere addition, and have identified genes that act in the suppression and formation of GCRs. Insights from these studies have contributed to the understanding of pathways and mechanisms that suppress genome instability and how these pathways cooperate with each other. Integrated models for the formation and suppression of GCRs are discussed.


Assuntos
Genoma Fúngico , Instabilidade Genômica , Saccharomyces cerevisiae/genética , Aberrações Cromossômicas , Rearranjo Gênico
12.
Proc Natl Acad Sci U S A ; 114(14): 3607-3612, 2017 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-28265089

RESUMO

Mammalian and Saccharomyces cerevisiae mismatch repair (MMR) proteins catalyze two MMR reactions in vitro. In one, mispair binding by either the MutS homolog 2 (Msh2)-MutS homolog 6 (Msh6) or the Msh2-MutS homolog 3 (Msh3) stimulates 5' to 3' excision by exonuclease 1 (Exo1) from a single-strand break 5' to the mispair, excising the mispair. In the other, Msh2-Msh6 or Msh2-Msh3 activate the MutL homolog 1 (Mlh1)-postmeiotic segregation 1 (Pms1) endonuclease in the presence of a mispair and a nick 3' to the mispair, to make nicks 5' to the mispair, allowing Exo1 to excise the mispair. DNA polymerase δ (Pol δ) is thought to catalyze DNA synthesis to fill in the gaps resulting from mispair excision. However, colocalization of the S. cerevisiae mispair recognition proteins with the replicative DNA polymerases during DNA replication has suggested that DNA polymerase ε (Pol ε) may also play a role in MMR. Here we describe the reconstitution of Pol ε-dependent MMR using S. cerevisiae proteins. A mixture of Msh2-Msh6 (or Msh2-Msh3), Exo1, RPA, RFC-Δ1N, PCNA, and Pol ε was found to catalyze both short-patch and long-patch 5' nick-directed MMR of a substrate containing a +1 (+T) mispair. When the substrate contained a nick 3' to the mispair, a mixture of Msh2-Msh6 (or Msh2-Msh3), Exo1, RPA, RFC-Δ1N, PCNA, and Pol ε was found to catalyze an MMR reaction that required Mlh1-Pms1. These results demonstrate that Pol ε can act in eukaryotic MMR in vitro.


Assuntos
Reparo de Erro de Pareamento de DNA , DNA Polimerase Dirigida por DNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Proteína 1 Homóloga a MutL/metabolismo , Proteínas MutL/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Proteína 3 Homóloga a MutS/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
13.
Hum Mol Genet ; 26(2): 258-269, 2017 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-28013290

RESUMO

The integrity and dynamic properties of the microtubule cytoskeleton are indispensable for the development of the mammalian brain. Consequently, mutations in the genes that encode the structural component (the α/ß-tubulin heterodimer) can give rise to severe, sporadic neurodevelopmental disorders. These are commonly referred to as the tubulinopathies. Here we report the addition of recessive quadrupedalism, also known as Uner Tan syndrome (UTS), to the growing list of diseases caused by tubulin variants. Analysis of a consanguineous UTS family identified a biallelic TUBB2B mutation, resulting in a p.R390Q amino acid substitution. In addition to the identifying quadrupedal locomotion, all three patients showed severe cerebellar hypoplasia. None, however, displayed the basal ganglia malformations typically associated with TUBB2B mutations. Functional analysis of the R390Q substitution revealed that it did not affect the ability of ß-tubulin to fold or become assembled into the α/ß-heterodimer, nor did it influence the incorporation of mutant-containing heterodimers into microtubule polymers. The 390Q mutation in S. cerevisiae TUB2 did not affect growth under basal conditions, but did result in increased sensitivity to microtubule-depolymerizing drugs, indicative of a mild impact of this mutation on microtubule function. The TUBB2B mutation described here represents an unusual recessive mode of inheritance for missense-mediated tubulinopathies and reinforces the sensitivity of the developing cerebellum to microtubule defects.


Assuntos
Cerebelo/anormalidades , Malformações do Desenvolvimento Cortical/genética , Microtúbulos/genética , Malformações do Sistema Nervoso/genética , Tubulina (Proteína)/genética , Adulto , Substituição de Aminoácidos/genética , Gânglios da Base/patologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/patologia , Cerebelo/fisiopatologia , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/fisiopatologia , Feminino , Homozigoto , Humanos , Masculino , Malformações do Desenvolvimento Cortical/fisiopatologia , Microtúbulos/patologia , Mutação , Malformações do Sistema Nervoso/fisiopatologia , Fenótipo , Saccharomyces cerevisiae/genética
14.
PLoS Genet ; 12(8): e1006302, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27579875

RESUMO

[This corrects the article DOI: 10.1371/journal.pgen.1003670.].

15.
Nat Commun ; 7: 11256, 2016 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-27071721

RESUMO

Gross chromosomal rearrangements (GCRs) play an important role in human diseases, including cancer. The identity of all Genome Instability Suppressing (GIS) genes is not currently known. Here multiple Saccharomyces cerevisiae GCR assays and query mutations were crossed into arrays of mutants to identify progeny with increased GCR rates. One hundred eighty two GIS genes were identified that suppressed GCR formation. Another 438 cooperatively acting GIS genes were identified that were not GIS genes, but suppressed the increased genome instability caused by individual query mutations. Analysis of TCGA data using the human genes predicted to act in GIS pathways revealed that a minimum of 93% of ovarian and 66% of colorectal cancer cases had defects affecting one or more predicted GIS gene. These defects included loss-of-function mutations, copy-number changes associated with reduced expression, and silencing. In contrast, acute myeloid leukaemia cases did not appear to have defects affecting the predicted GIS genes.


Assuntos
Rearranjo Gênico/genética , Redes Reguladoras de Genes , Genoma Fúngico , Neoplasias/genética , Saccharomyces cerevisiae/genética , Cromossomos Fúngicos/genética , Elementos Facilitadores Genéticos/genética , Epistasia Genética , Genes Fúngicos , Instabilidade Genômica , Humanos , Mutação/genética
17.
J Biol Chem ; 290(35): 21580-90, 2015 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-26170454

RESUMO

Previous studies reported the reconstitution of an Mlh1-Pms1-independent 5' nick-directed mismatch repair (MMR) reaction using Saccharomyces cerevisiae proteins. Here we describe the reconstitution of a mispair-dependent Mlh1-Pms1 endonuclease activation reaction requiring Msh2-Msh6 (or Msh2-Msh3), proliferating cell nuclear antigen (PCNA), and replication factor C (RFC) and a reconstituted Mlh1-Pms1-dependent 3' nick-directed MMR reaction requiring Msh2-Msh6 (or Msh2-Msh3), exonuclease 1 (Exo1), replication protein A (RPA), RFC, PCNA, and DNA polymerase δ. Both reactions required Mg(2+) and Mn(2+) for optimal activity. The MMR reaction also required two reaction stages in which the first stage required incubation of Mlh1-Pms1 with substrate DNA, with or without Msh2-Msh6 (or Msh2-Msh3), PCNA, and RFC but did not require nicking of the substrate, followed by a second stage in which other proteins were added. Analysis of different mutant proteins demonstrated that both reactions required a functional Mlh1-Pms1 endonuclease active site, as well as mispair recognition and Mlh1-Pms1 recruitment by Msh2-Msh6 but not sliding clamp formation. Mutant Mlh1-Pms1 and PCNA proteins that were defective for Exo1-independent but not Exo1-dependent MMR in vivo were partially defective in the Mlh1-Pms1 endonuclease and MMR reactions, suggesting that both reactions reflect the activation of Mlh1-Pms1 seen in Exo1-independent MMR in vivo. The availability of this reconstituted MMR reaction should now make it possible to better study both Exo1-independent and Exo1-dependent MMR.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Reparo de Erro de Pareamento de DNA , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Biocatálise/efeitos dos fármacos , Cátions Bivalentes/farmacologia , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Genes Dominantes , Proteína 1 Homóloga a MutL , Proteínas MutL , Proteínas Mutantes/metabolismo , Mutação , Saccharomyces cerevisiae/efeitos dos fármacos
18.
DNA Repair (Amst) ; 32: 24-32, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25956862

RESUMO

DNA mismatch repair (MMR) acts to repair mispaired bases resulting from misincorporation errors during DNA replication and also recognizes mispaired bases in recombination (HR) intermediates. Exonuclease 1 (Exo1) is a 5' → 3' exonuclease that participates in a number of DNA repair pathways. Exo1 was identified as an exonuclease that participates in Saccharomyces cerevisiae and human MMR where it functions to excise the daughter strand after mispair recognition, and additionally Exo1 functions in end resection during HR. However, Exo1 is not absolutely required for end resection during HR in vivo. Similarly, while Exo1 is required in MMR reactions that have been reconstituted in vitro, genetics studies have shown that it is not absolutely required for MMR in vivo suggesting the existence of Exo1-independent and Exo1-dependent MMR subpathways. Here, we review what is known about the Exo1-independent and Exo1-dependent subpathways, including studies of mutations in MMR genes that specifically disrupt either subpathway.


Assuntos
Reparo de Erro de Pareamento de DNA , DNA Fúngico/química , Exodesoxirribonucleases/química , Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae/enzimologia , Pareamento Incorreto de Bases , Quebras de DNA de Cadeia Dupla , Replicação do DNA , DNA Fúngico/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Humanos , Modelos Moleculares , Mutação , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética
19.
Chromosoma ; 124(4): 443-62, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25862369

RESUMO

The genome of all organisms is constantly being challenged by endogenous and exogenous sources of DNA damage. Errors like base:base mismatches or small insertions and deletions, primarily introduced by DNA polymerases during DNA replication are repaired by an evolutionary conserved DNA mismatch repair (MMR) system. The MMR system, together with the DNA replication machinery, promote repair by an excision and resynthesis mechanism during or after DNA replication, increasing replication fidelity by up-to-three orders of magnitude. Consequently, inactivation of MMR genes results in elevated mutation rates that can lead to increased cancer susceptibility in humans. In this review, we summarize our current understanding of MMR with a focus on the different MMR protein complexes, their function and structure. We also discuss how recent findings have provided new insights in the spatio-temporal regulation and mechanism of MMR.


Assuntos
Reparo de Erro de Pareamento de DNA , Replicação do DNA , Eucariotos , Humanos
20.
PLoS One ; 9(9): e108123, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25247923

RESUMO

The absence of Tsa1, a key peroxiredoxin that scavenges H2O2 in Saccharomyces cerevisiae, causes the accumulation of a broad spectrum of mutations. Deletion of TSA1 also causes synthetic lethality in combination with mutations in RAD51 or several key genes involved in DNA double-strand break repair. In the present study, we propose that the accumulation of reactive oxygen species (ROS) is the primary cause of genome instability of tsa1Δ cells. In searching for spontaneous suppressors of synthetic lethality of tsa1Δ rad51Δ double mutants, we identified that the loss of thioredoxin reductase Trr1 rescues their viability. The trr1Δ mutant displayed a Can(R) mutation rate 5-fold lower than wild-type cells. Additional deletion of TRR1 in tsa1Δ mutant reduced substantially the Can(R) mutation rate of tsa1Δ strain (33-fold), and to a lesser extent, of rad51Δ strain (4-fold). Loss of Trr1 induced Yap1 nuclear accumulation and over-expression of a set of Yap1-regulated oxido-reductases with antioxidant properties that ultimately re-equilibrate intracellular redox environment, reducing substantially ROS-associated DNA damages. This trr1Δ -induced effect was largely thioredoxin-dependent, probably mediated by oxidized forms of thioredoxins, the primary substrates of Trr1. Thioredoxin Trx1 and Trx2 were constitutively and strongly oxidized in the absence of Trr1. In trx1Δ trx2Δ cells, Yap1 was only moderately activated; consistently, the trx1Δ trx2Δ double deletion failed to efficiently rescue the viability of tsa1Δ rad51Δ. Finally, we showed that modulation of the dNTP pool size also influences the formation of spontaneous mutation in trr1Δ and trx1Δ trx2Δ strains. We present a tentative model that helps to estimate the respective impact of ROS level and dNTP concentration in the generation of spontaneous mutations.


Assuntos
Instabilidade Genômica , Mutação , Peroxidases/genética , Proteínas de Saccharomyces cerevisiae/genética , Tiorredoxina Redutase 1/genética , Dano ao DNA/genética , Reparo do DNA/genética , Peroxidases/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Tiorredoxina Redutase 1/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
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