Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 16 de 16
Filtrar
1.
J Biol Chem ; 300(7): 107430, 2024 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-38825008

RESUMO

The nuclear envelope (NE) is a permeable barrier that maintains nuclear-cytoplasmic compartmentalization and ensures nuclear function; however, it ruptures in various situations such as mechanical stress and mitosis. Although the protein components for sealing a ruptured NE have been identified, the mechanism by which lipid components are involved in this process remains to be elucidated. Here, we found that an inner nuclear membrane (INM) protein Bqt4 directly interacts with phosphatidic acid (PA) and serves as a platform for NE maintenance in the fission yeast Schizosaccharomyces pombe. The intrinsically disordered region (IDR) of Bqt4, proximal to the transmembrane domain, binds to PA and forms a solid aggregate in vitro. Excessive accumulation of Bqt4 IDR in INM results in membrane overproliferation and lipid droplet formation in the nucleus, leading to centromere dissociation from the NE and chromosome missegregation. Our findings suggest that Bqt4 IDR controls nuclear membrane homeostasis by recruiting PA to the INM, thereby maintaining the structural integrity of the NE.

2.
J Cell Sci ; 136(10)2023 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-37078207

RESUMO

Maintaining the integrity of the nuclear envelope (NE) is essential for preventing genomic DNA damage. Recent studies have shown that enzymes that catalyze lipid synthesis are involved in NE maintenance, but the underlying mechanism remains unclear. Here, we found that the ceramide synthase (CerS) homolog in the fission yeast Schizosaccharomyces pombe Tlc4 (SPAC17A2.02c) suppressed NE defects in cells lacking the NE proteins Lem2 and Bqt4. Tlc4 possesses a TRAM/LAG1/CLN8 domain that is conserved in CerS proteins and functions through its non-catalytic activity. Tlc4 was localized at the NE and endoplasmic reticulum, similar to CerS proteins, and also showed unique additional localization at the cis- and medial-Golgi cisternae. Growth and mutation analyses revealed that Golgi localization of Tlc4 was tightly linked to its activity of suppressing the defects in the double-deletion mutant of Lem2 and Bqt4. Our results suggest that Lem2 and Bqt4 control the translocation of Tlc4 from the NE to the Golgi, which is necessary for maintaining NE integrity.


Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Membrana Nuclear/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas Nucleares/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo
3.
Nucleic Acids Res ; 44(1): 232-44, 2016 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-26433224

RESUMO

The centromeres of many eukaryotic chromosomes are established epigenetically on potentially variable tandem repeats; hence, these chromosomes are at risk of being acentric. We reported previously that artificially created acentric chromosomes in the fission yeast Schizosaccharomyces pombe can be rescued by end-to-end fusion with functional chromosomes. Here, we show that most acentric/functional chromosome fusion events in S. pombe cells harbouring an acentric chromosome I differed from the non-homologous end-joining-mediated rearrangements that result in deleterious dicentric fusions in normal cells, and were elicited by a previously unidentified homologous recombination (HR) event between chromosome end-associated sequences. The subtelomere repeats associated with the non-fusogenic ends were also destabilized in the surviving cells, suggesting a causal link between general subtelomere destabilization and acentric/functional chromosome fusion. A mutational analysis indicated that a non-canonical HR pathway was involved in the rearrangement. These findings are indicative of a latent mechanism that conditionally induces general subtelomere instability, presumably in the face of accidental centromere loss events, resulting in rescue of the fatal acentric chromosomes by interchromosomal HR.


Assuntos
Cromossomos Fúngicos , Rearranjo Gênico , Recombinação Homóloga , Telômero , Mapeamento Cromossômico , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Instabilidade Genômica , Modelos Biológicos , Rad51 Recombinase/deficiência , Schizosaccharomyces/genética
4.
Nature ; 457(7229): 612-5, 2009 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-19079240

RESUMO

In nature, organisms are exposed to chronic low-dose ultraviolet light (CLUV) as opposed to the acute high doses common to laboratory experiments. Analysis of the cellular response to acute high-dose exposure has delineated the importance of direct DNA repair by the nucleotide excision repair pathway and for checkpoint-induced cell cycle arrest in promoting cell survival. Here we examine the response of yeast cells to CLUV and identify a key role for the RAD6-RAD18-RAD5 error-free postreplication repair (RAD6 error-free PRR) pathway in promoting cell growth and survival. We show that loss of the RAD6 error-free PRR pathway results in DNA-damage-checkpoint-induced G2 arrest in CLUV-exposed cells, whereas wild-type and nucleotide-excision-repair-deficient cells are largely unaffected. Cell cycle arrest in the absence of the RAD6 error-free PRR pathway was not caused by a repair defect or by the accumulation of ultraviolet-induced photoproducts. Notably, we observed increased replication protein A (RPA)- and Rad52-yellow fluorescent protein foci in the CLUV-exposed rad18Delta cells and demonstrated that Rad52-mediated homologous recombination is required for the viability of the rad18Delta cells after release from CLUV-induced G2 arrest. These and other data presented suggest that, in response to environmental levels of ultraviolet exposure, the RAD6 error-free PRR pathway promotes replication of damaged templates without the generation of extensive single-stranded DNA regions. Thus, the error-free PRR pathway is specifically important during chronic low-dose ultraviolet exposure to prevent counter-productive DNA checkpoint activation and allow cells to proliferate normally.


Assuntos
Adenosina Trifosfatases/metabolismo , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos da radiação , Enzimas de Conjugação de Ubiquitina/metabolismo , Raios Ultravioleta , Adenosina Trifosfatases/deficiência , Adenosina Trifosfatases/genética , Dano ao DNA , DNA Helicases , Replicação do DNA/efeitos da radiação , DNA Fúngico/efeitos da radiação , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Fase G2/efeitos da radiação , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinação Genética , Proteína de Replicação A/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Enzimas de Conjugação de Ubiquitina/deficiência , Enzimas de Conjugação de Ubiquitina/genética
5.
J Biol Chem ; 288(41): 29229-37, 2013 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-23974212

RESUMO

Escherichia coli RecN is an SMC (structural maintenance of chromosomes) family protein that is required for DNA double-strand break (DSB) repair. Previous studies show that GFP-RecN forms nucleoid-associated foci in response to DNA damage, but the mechanism by which RecN is recruited to the nucleoid is unknown. Here, we show that the assembly of GFP-RecN foci on the nucleoid in response to DNA damage involves a functional interaction between RecN and RecA. A novel RecA allele identified in this work, recA(Q300R), is proficient in SOS induction and repair of UV-induced DNA damage, but is deficient in repair of mitomycin C (MMC)-induced DNA damage. Cells carrying recA(Q300R) fail to recruit RecN to DSBs and accumulate fragmented chromosomes after exposure to MMC. The ATPase-deficient RecN(K35A) binds and forms foci at MMC-induced DSBs, but is not released from the MMC-induced DNA lesions, resulting in a defect in homologous recombination-dependent DSB repair. These data suggest that RecN plays a crucial role in homologous recombination-dependent DSB repair and that it is required upstream of RecA-mediated strand exchange.


Assuntos
Proteínas de Bactérias/metabolismo , Quebras de DNA de Cadeia Dupla , Enzimas de Restrição do DNA/metabolismo , Escherichia coli/metabolismo , Recombinases Rec A/metabolismo , Alquilantes/farmacologia , Proteínas de Bactérias/genética , Dano ao DNA , Enzimas de Restrição do DNA/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência , Mitomicina/farmacologia , Mutação de Sentido Incorreto , Recombinases Rec A/genética , Reparo de DNA por Recombinação/genética , Resposta SOS em Genética/genética
6.
Nucleic Acids Res ; 40(17): 8406-15, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-22743272

RESUMO

UV radiation induces two major types of DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidine photoproducts, which are both primarily repaired by nucleotide excision repair (NER). Here, we investigated how chronic low-dose UV (CLUV)-induced mutagenesis occurs in rad14Δ NER-deficient yeast cells, which lack the yeast orthologue of human xeroderma pigmentosum A (XPA). The results show that rad14Δ cells have a marked increase in CLUV-induced mutations, most of which are C→T transitions in the template strand for transcription. Unexpectedly, many of the CLUV-induced C→T mutations in rad14Δ cells are dependent on translesion synthesis (TLS) DNA polymerase η, encoded by RAD30, despite its previously established role in error-free TLS. Furthermore, we demonstrate that deamination of cytosine-containing CPDs contributes to CLUV-induced mutagenesis. Taken together, these results uncover a novel role for Polη in the induction of C→T transitions through deamination of cytosine-containing CPDs in CLUV-exposed NER deficient cells. More generally, our data suggest that Polη can act as both an error-free and a mutagenic DNA polymerase, depending on whether the NER pathway is available to efficiently repair damaged templates.


Assuntos
Reparo do DNA , DNA Polimerase Dirigida por DNA/fisiologia , Mutagênese , Raios Ultravioleta , Canavanina/farmacologia , Dano ao DNA , Enzimas Reparadoras do DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Desaminação , Deleção de Genes , Dímeros de Pirimidina/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/genética
7.
J Biochem ; 174(1): 33-46, 2023 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-36799444

RESUMO

The nuclear envelope (NE) is a double-membrane structure consisting of inner and outer membranes that spatially separate the nucleus from the cytoplasm, and its function is critical for cellular functions such as genome maintenance. In the fission yeast, Schizosaccharomyces pombe, the inner nuclear membrane proteins, Lem2 and Bqt4, play pivotal roles in maintaining the NE structure. We previously found that the double deletion of lem2+ and bqt4+ causes a synthetic lethal defect associated with severe NE rupture, and overexpression of Elo2, a solo very-long-chain fatty acid elongase, suppresses this defect by restoring the NE. However, the molecular basis of this restoration remains elusive. To address this, we identified Lem2- and Bqt4-binding proteins via immunoprecipitation and mass spectrometry in this study. Forty-five and 23 proteins were identified as Lem2- and Bqt4-binding proteins, respectively. Although these binding proteins partially overlapped, Lem2 and Bqt4 interacted with different types of lipid metabolic enzymes: Cho2, Ole1 and Erg11 for Lem2 and Cwh43 for Bqt4. These enzymes are known to be involved in various lipid synthesis processes, suggesting that Lem2 and Bqt4 may contribute to the regulation of lipid synthesis by binding to these enzymes.


Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Schizosaccharomyces/metabolismo , Membrana Nuclear/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Membrana/metabolismo , Lipídeos
8.
Commun Biol ; 3(1): 276, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483293

RESUMO

The nuclear envelope (NE) continues to the endoplasmic reticulum (ER). Proper partitioning of NE and ER is crucial for cellular activity, but the key factors maintaining the boundary between NE and ER remain to be elucidated. Here we show that the conserved membrane proteins Lem2 and Lnp1 cooperatively play a crucial role in maintaining the NE-ER membrane boundary in fission yeast Schizosaccharomyces pombe. Cells lacking both Lem2 and Lnp1 caused severe growth defects associated with aberrant expansion of the NE/ER membranes, abnormal leakage of nuclear proteins, and abnormal formation of vacuolar-like structures in the nucleus. Overexpression of the ER membrane protein Apq12 rescued the growth defect associated with membrane disorder caused by the loss of Lem2 and Lnp1. Genetic analysis showed that Apq12 had overlapping functions with Lnp1. We propose that a membrane protein network with Lem2 and Lnp1 acts as a critical factor to maintain the NE-ER boundary.


Assuntos
Proteínas de Ligação a DNA/genética , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/genética , Membrana Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo
9.
Genetics ; 180(1): 41-50, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18757937

RESUMO

The ESC2 gene encodes a protein with two tandem C-terminal SUMO-like domains and is conserved from yeasts to humans. Previous studies have implicated Esc2 in gene silencing. Here, we explore the functional significance of SUMO-like domains and describe a novel role for Esc2 in promoting genome integrity during DNA replication. This study shows that esc2Delta cells are modestly sensitive to hydroxyurea (HU) and defective in sister chromatid cohesion and have a reduced life span, and these effects are enhanced by deletion of the RRM3 gene that is a Pif1-like DNA helicase. esc2Delta rrm3Delta cells also have a severe growth defect and accumulate DNA damage in late S/G2. In contrast, esc2Delta does not enhance the HU sensitivity or sister chromatid cohesion defect in mrc1Delta cells, but rather partially suppresses both phenotypes. We also show that deletion of both Esc2 SUMO-like domains destabilizes Esc2 protein and functionally inactivates Esc2, but this phenotype is suppressed by an Esc2 variant with an authentic SUMO domain. These results suggest that Esc2 is functionally equivalent to a stable SUMO fusion protein and plays important roles in facilitating DNA replication fork progression and sister chromatid cohesion that would otherwise impede the replication fork in rrm3Delta cells.


Assuntos
Cromátides/genética , Genoma Fúngico , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Ciclo Celular , Proteínas de Ciclo Celular , Segregação de Cromossomos , Dano ao DNA , Replicação do DNA , Deleção de Genes , Inativação Gênica , Hidroxiureia/farmacologia , Modelos Genéticos , Plasmídeos/metabolismo , Proteínas Recombinantes de Fusão/química , Técnicas do Sistema de Duplo-Híbrido
10.
Mol Cell Biol ; 26(14): 5509-17, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16809783

RESUMO

Proliferating cell nuclear antigen (PCNA), a sliding clamp required for processive DNA synthesis, provides attachment sites for various other proteins that function in DNA replication, DNA repair, cell cycle progression and chromatin assembly. It has been shown that differential posttranslational modifications of PCNA by ubiquitin or SUMO play a pivotal role in controlling the choice of pathway for rescuing stalled replication forks. Here, we explored the roles of Mgs1 and PCNA in replication fork rescue. We provide evidence that Mgs1 physically associates with PCNA and that Mgs1 helps suppress the RAD6 DNA damage tolerance pathway in the absence of exogenous DNA damage. We also show that PCNA sumoylation inhibits the growth of mgs1 rad18 double mutants, in which PCNA sumoylation and the Srs2 DNA helicase coordinately prevent RAD52-dependent homologous recombination. The proposed roles for Mgs1, Srs2, and modified PCNA during replication arrest highlight the importance of modulating the RAD6 and RAD52 pathways to avoid genome instability.


Assuntos
Adenosina Trifosfatases/metabolismo , Dano ao DNA , DNA Helicases/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Adenosina Trifosfatases/genética , Sequência de Bases , DNA Helicases/genética , Replicação do DNA , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes Fúngicos , Instabilidade Genômica , Lisina/química , Mutação , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/genética , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética
11.
Nat Struct Mol Biol ; 20(12): 1397-406, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24186062

RESUMO

In most eukaryotes, centromeres are epigenetically defined by nucleosomes that contain the histone H3 variant centromere protein A (CENP-A). Specific targeting of the CENP-A-loading chaperone to the centromere is vital for stable centromere propagation; however, the existence of ectopic centromeres (neocentromeres) indicates that this chaperone can function in different chromatin environments. The mechanism responsible for accommodating the CENP-A chaperone at noncentromeric regions is poorly understood. Here, we report the identification of transient, immature neocentromeres in Schizosaccharomyces pombe that show reduced association with the CENP-A chaperone Scm3, owing to persistence of the histone H2A variant H2A.Z. After the acquisition of adjacent heterochromatin or relocation of the immature neocentromeres to subtelomeric regions, H2A.Z was depleted and Scm3 was replenished, thus leading to subsequent stabilization of the neocentromeres. These findings provide new insights into histone variant-mediated epigenetic control of neocentromere establishment.


Assuntos
Centrômero/fisiologia , Epigenômica , Histonas/metabolismo , Modelos Genéticos , Schizosaccharomyces/genética , Autoantígenos/química , Autoantígenos/metabolismo , Autoantígenos/fisiologia , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Centrômero/metabolismo , Centrômero/ultraestrutura , Proteína Centromérica A , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/fisiologia , Histonas/fisiologia , Nucleossomos/metabolismo , Schizosaccharomyces/metabolismo , Schizosaccharomyces/ultraestrutura , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/fisiologia
12.
Mol Cell Biol ; 30(20): 4840-50, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20713444

RESUMO

Differential posttranslational modification of proliferating cell nuclear antigen (PCNA) by ubiquitin or SUMO plays an important role in coordinating the processes of DNA replication and DNA damage tolerance. Previously it was shown that the loss of RAD6-dependent error-free postreplication repair (PRR) results in DNA damage checkpoint-mediated G(2) arrest in cells exposed to chronic low-dose UV radiation (CLUV), whereas wild-type and nucleotide excision repair-deficient cells are largely unaffected. In this study, we report that suppression of homologous recombination (HR) in PRR-deficient cells by Srs2 and PCNA sumoylation is required for checkpoint activation and checkpoint maintenance during CLUV irradiation. Cyclin-dependent kinase (CDK1)-dependent phosphorylation of Srs2 did not influence checkpoint-mediated G(2) arrest or maintenance in PRR-deficient cells but was critical for HR-dependent checkpoint recovery following release from CLUV exposure. These results indicate that Srs2 plays an important role in checkpoint-mediated reversible G(2) arrest in PRR-deficient cells via two separate HR-dependent mechanisms. The first (required to suppress HR during PRR) is regulated by PCNA sumoylation, whereas the second (required for HR-dependent recovery following CLUV exposure) is regulated by CDK1-dependent phosphorylation.


Assuntos
DNA Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Proteína Quinase CDC2/metabolismo , DNA Helicases/genética , Reparo do DNA , Replicação do DNA , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fase G2/efeitos da radiação , Deleção de Genes , Genes Fúngicos , Modelos Biológicos , Mutação , Fosforilação , Antígeno Nuclear de Célula em Proliferação/metabolismo , Tolerância a Radiação/genética , Tolerância a Radiação/fisiologia , Recombinação Genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Raios Ultravioleta
13.
J Biol Chem ; 281(41): 30941-6, 2006 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-16914543

RESUMO

Protein degradation in bacteria plays a dynamic and critical role in the cellular response to environmental stimuli such as heat shock and DNA damage and in removing damaged proteins or protein aggregates. Escherichia coli recN is a member of the structural maintenance of chromosomes family and is required for DNA double strand break (DSB) repair. This study shows that RecN protein has a short half-life and its degradation is dependent on the cytoplasmic protease ClpXP and a degradation signal at the C terminus of RecN. In cells with DNA DSBs, green fluorescent protein-RecN localized in discrete foci on nucleoids and formed visible aggregates in the cytoplasm, both of which disappeared rapidly in wild-type cells when DSBs were repaired. In contrast, in DeltaclpX cells, RecN aggregates persisted in the cytoplasm after release from DNA damage. Furthermore, analysis of cells experiencing chronic DNA damage revealed that proteolytic removal of RecN aggregates by ClpXP was important for cell viability. These data demonstrate that ClpXP is a critical factor in the cellular clearance of cytoplasmic RecN aggregates from the cell and therefore plays an important role in DNA damage tolerance.


Assuntos
Proteínas de Bactérias/química , Dano ao DNA , Enzimas de Restrição do DNA/química , Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Proteínas de Bactérias/metabolismo , Citoplasma/enzimologia , Citoplasma/metabolismo , Reparo do DNA , Enzimas de Restrição do DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência , Mitomicina/farmacologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Fatores de Tempo
14.
Genes Cells ; 10(3): 181-91, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15743409

RESUMO

Escherichia coli RecA protein plays a role in DNA homologous recombination, recombination repair, and the rescue of stalled or collapsed replication forks. The mgsA (rarA) gene encodes a highly conserved DNA-dependent ATPase, whose yeast orthologue, MGS1, plays a role in maintaining genomic stability. In this study, we show a functional relationship between mgsA and recA during DNA replication. The mgsA recA double mutant grows more slowly and has lower viability than a recA single mutant, but they are equally sensitive to UV-induced DNA damage. Mutations in mgsA and recA cause lethality in DNA polymerase I deficient cells, and suppress the temperature-dependent growth defect of dnaE486 (Pol III alpha-catalytic subunit). Moreover, recAS25P, a novel recA allele identified in this work, does not complement the slow growth of DeltamgsA DeltarecA cells or the lethality of polA12 DeltarecA, but is proficient in DNA repair, homologous recombination, SOS mutagenesis and SOS induction. These results suggest that RecA and MgsA are functionally redundant in rescuing stalled replication forks, and that the DNA repair and homologous recombination functions of RecA are separated from its function to maintain progression of replication fork.


Assuntos
Adenosina Trifosfatases/metabolismo , DNA Helicases/metabolismo , Replicação do DNA/fisiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Recombinases Rec A/metabolismo , Adenosina Trifosfatases/genética , DNA Helicases/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Mutação , Recombinases Rec A/genética
15.
Genes Dev ; 18(15): 1886-97, 2004 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-15289460

RESUMO

The RecQ protein family is a highly conserved group of DNA helicases that play roles in maintaining genomic stability. In this study, we present biochemical and genetic evidence that Escherichia coli RecQ processes stalled replication forks and participates in SOS signaling. Cells that carry dnaE486, a mutation in the DNA polymerase III alpha-catalytic subunit, induce an RecA-dependent SOS response and become highly filamented at the semirestrictive temperature (38 degrees C). An recQ mutation suppresses the induction of SOS response and the filamentation in the dnaE486 mutant at 38 degrees C, causing appearance of a high proportion of anucleate cells. In vitro, RecQ binds and unwinds forked DNA substrates with a gap on the leading strand more efficiently than those with a gap on the lagging strand or Holliday junction DNA. RecQ unwinds the template duplex ahead of the fork, and then the lagging strand is unwound. Consequently, this process generates a single-stranded DNA (ssDNA) gap on the lagging strand adjacent to a replication fork. These results suggest that RecQ functions to generate an initiating signal that can recruit RecA for SOS induction and recombination at stalled replication forks, which are required for the cell cycle checkpoint and resumption of DNA replication.


Assuntos
Adenosina Trifosfatases/fisiologia , DNA Helicases/fisiologia , Replicação do DNA , Escherichia coli/enzimologia , Genoma Bacteriano , Transdução de Sinais/fisiologia , Proteínas Son Of Sevenless/metabolismo , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Escherichia coli/genética , Mutação/genética , RecQ Helicases , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura
16.
Lancet ; 361(9359): 743-9, 2003 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-12620739

RESUMO

BACKGROUND: Vibrio parahaemolyticus, a gram-negative marine bacterium, is a worldwide cause of food-borne gastroenteritis. V parahaemolyticus strains of a few specific serotypes, probably derived from a common clonal ancestor, have lately caused a pandemic of gastroenteritis. The organism is phylogenetically close to V cholerae, the causative agent of cholera. METHODS: The whole genome sequence of a clinical V parahaemolyticus strain RIMD2210633 was established by shotgun sequencing. The coding sequences were identified by use of Gambler and Glimmer programs. Comparative analysis with the V cholerae genome was undertaken with MUMmer. FINDINGS: The genome consisted of two circular chromosomes of 3288558 bp and 1877212 bp; it contained 4832 genes. Comparison of the V parahaemolyticus genome with that of V cholerae showed many rearrangements within and between the two chromosomes. Genes for the type III secretion system (TTSS) were identified in the genome of V parahaemolyticus; V cholerae does not have these genes. INTERPRETATION: The TTSS is a central virulence factor of diarrhoea-causing bacteria such as shigella, salmonella, and enteropathogenic Escherichia coli, which cause gastroenteritis by invading or intimately interacting with intestinal epithelial cells. Our results suggest that V parahaemolyticus and V cholerae use distinct mechanisms to establish infection. This finding explains clinical features of V parahaemolyticus infections, which commonly include inflammatory diarrhoea and in some cases systemic manifestations such as septicaemia, distinct from those of V cholerae infections, which are generally associated with non-inflammatory diarrhoea.


Assuntos
Cólera/microbiologia , DNA Bacteriano/genética , Doenças Transmitidas por Alimentos/microbiologia , Gastroenterite/microbiologia , Vibrio cholerae/genética , Vibrio parahaemolyticus/genética , Mapeamento Cromossômico , DNA Circular/genética , Humanos , Dados de Sequência Molecular , Análise de Sequência de DNA , Sorotipagem , Vibrio cholerae/patogenicidade , Vibrio parahaemolyticus/patogenicidade , Virulência/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA