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1.
Fungal Genet Biol ; 49(4): 263-70, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22395065

RESUMO

Stalled replication forks easily collapse and such structures can induce DNA strand breaks or toxic recombination products. Therefore, factors involved in stabilization of replication should be important for genome integrity. In our previous study, loss of both ATM and ATR homologues was shown to cause growth defects and chromosome instability in Neurospora crassa. To elucidate the relationships between these defects and replication instability, we focused on one of the viable replication factors, mrc1. We identified an mrc1 homologue from the N. crassa genome database. The mrc1 disruptant was sensitive to the replication inhibitor hydroxyurea (HU) and delayed restart of the cell cycle from HU treatment. Importantly, HU treatment induced histone H2A phosphorylation in the mrc1 mutant but not in the wild type. Furthermore, the HU-induced H2A phosphorylation was completely dependent on the ATM homologue mus-21, and dysfunction of mus-21 increased HU sensitivity of the mrc1 mutant. These results indicate that Neurospora mrc1 is important for stabilization of replication forks and that loss of mrc1 causes activation of the DNA damage checkpoint. Unexpectedly, loss of mrc1 did not affect cell growth, but the deletion of mrc1 reduced hyphal growth speed and conidia viability in the mus-9 and mus-21 mutants. The mrc1 mus-9 and mrc1 mus-21 double mutants also showed accumulation of micronuclei, which is a typical marker of chromosome instability. These results imply that activation of the checkpoint pathway can protect cells from instability of DNA replication caused by loss of mrc1.


Assuntos
Instabilidade Cromossômica/genética , Replicação do DNA/genética , DNA Fúngico/genética , Proteínas Fúngicas/genética , Neurospora crassa/fisiologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Instabilidade Cromossômica/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Replicação do DNA/efeitos dos fármacos , Proteínas Fúngicas/metabolismo , Histonas/efeitos dos fármacos , Histonas/genética , Histonas/metabolismo , Hidroxiureia/farmacologia , Hifas/genética , Hifas/crescimento & desenvolvimento , Micronúcleos com Defeito Cromossômico , Mutação , Neurospora crassa/citologia , Neurospora crassa/efeitos dos fármacos , Neurospora crassa/genética , Inibidores da Síntese de Ácido Nucleico/farmacologia , Fosforilação/efeitos dos fármacos , Esporos Fúngicos/citologia , Esporos Fúngicos/genética , Esporos Fúngicos/fisiologia
2.
Fungal Genet Biol ; 47(10): 809-17, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20553930

RESUMO

Genome integrity is maintained by many cellular mechanisms in eukaryotes. One such mechanism functions during the cell cycle and is known as the DNA damage checkpoint. In the filamentous fungus Neurospora crassa, mus-9 and mus-21 are homologes of two key factors of the mammalian DNA damage checkpoint, ATR and ATM, respectively. We previously showed that mus-9 and mus-21 mutants are sensitive to DNA damage and that each mutant shows a characteristic growth defect: conidia from the mus-9 mutant have reduced viability and the mus-21 mutant exhibits slow hyphal growth. However, the relationship between these two genes has not been determined because strains carrying both mus-9 and mus-21 mutations could not be obtained. To facilitate analysis of a strain deficient in both mus-9 and mus-21, we introduced a specific mutation to the kinase domain of MUS-9 to generate a temperature-sensitive mus-9 allele (mus-9(ts)) which shows increased mutagen sensitivity at 37 degrees C. Then we crossed this strain with a mus-21 mutant to obtain a mus-9(ts) mus-21 double mutant. Growth of the mus-9(ts) mus-21 double mutant did not progress at the restrictive temperature (37 degrees C). Even at the permissive temperature (25 degrees C), this strain exhibited a higher mutagen sensitivity than that of the mus-9 and mus-21 single mutants, as well as slow hyphal growth and low viability of conidia. These results indicate that the mus-9(ts) mutation causes hypomorphic phenotypes in the mus-21 mutant and that these two genes regulate different pathways. Interestingly, we observed accumulation of micronuclei in the conidia of this double mutant, and such micronuclei were likely to correlate with spontaneous DSBs. Our results suggest that both mus-9 and mus-21 pathways are involved in DNA damage response, normal growth and maintenance of chromosome integrity, and that at least one of the pathways must be functional for survival.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Cromossomos Fúngicos/metabolismo , Proteínas de Ligação a DNA/fisiologia , Proteínas Fúngicas/fisiologia , Neurospora crassa/crescimento & desenvolvimento , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Sequência de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cromossomos Fúngicos/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Dados de Sequência Molecular , Mutação , Neurospora crassa/enzimologia , Neurospora crassa/genética , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína/genética , Alinhamento de Sequência , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética
3.
DNA Repair (Amst) ; 7(12): 1951-61, 2008 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-18790091

RESUMO

DNA damage checkpoint is an important mechanism for organisms to maintain genome integrity. In Neurospora crassa, mus-9 and mus-21 are homologues of ATR and ATM, respectively, which are pivotal factors of DNA damage checkpoint in mammals. A N. crassa clock gene prd-4 has been identified as a CHK2 homologue, but its role in DNA damage response had not been elucidated. In this study, we identified another CHK2 homologue and one CHK1 homologue from the N. crassa genome database. As disruption of these genes affected mutagen tolerance, we named them mus-59 and mus-58, respectively. The mus-58 mutant was sensitive to hydroxyurea (HU), but the mus-59 and prd-4 mutants showed the same HU sensitivity as that of the wild-type strain. This indicates the possibility that MUS-58 is involved in replication checkpoint and stabilization of stalled forks like mammalian CHK1. Phosphorylation of MUS-58 and MUS-59 was observed in the wild-type strain in response to mutagen treatments. Genetic relationships between those three genes and mus-9 or mus-21 indicated that the mus-9 mutation was epistatic to mus-58, and mus-21 was epistatic to prd-4. These relationships correspond to two signal pathways, ATR-CHK1 and ATM-CHK2 that have been established in mammalian cells. However, both the mus-9 mus-59 and mus-21 mus-58 double mutants showed an intermediate level between the two parental strains for CPT sensitivity. Furthermore, these double mutants showed severe growth defects. Our findings suggest that the DNA damage checkpoint of N. crassa is controlled by unique mechanisms.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Neurospora crassa/metabolismo , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Western Blotting , Proteínas de Ciclo Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2 , Ensaio de Unidades Formadoras de Colônias , Dano ao DNA/efeitos dos fármacos , Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA/efeitos dos fármacos , Proteínas Fúngicas/genética , Imunoprecipitação , Mutagênicos/farmacologia , Mutação/genética , Neurospora crassa/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo
4.
DNA Repair (Amst) ; 7(2): 213-29, 2008 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-17983847

RESUMO

The mutagen sensitive uvs-3 and mus-9 mutants of Neurospora show mutagen and hydroxyurea sensitivity, mutator effects and duplication instability typical of recombination repair and DNA damage checkpoint defective mutants. To determine the nature of these genes we used cosmids from a genomic library to clone the uvs-3 gene by complementation for MMS sensitivity. Mutation induction by transposon insertion and RIP defined the coding sequence. RFLP analysis confirmed that this sequence maps in the area of uvs-3 at the left telomere of LG IV. Analysis of the cDNA showed that the UVS-3 protein contains an ORF of 969 amino acids with one intron. It is homologous to UvsD of Aspergillus nidulans, a member of the ATRIP family of checkpoint proteins. It retains the N' terminal coiled-coil motif followed by four basic amino acids typical of these proteins and shows the highest homology in this region. The uvsD cDNA partially complements the defects of the uvs-3 mutation. The uvs-3 mutant shows a higher level of micronuclei in conidia and failure to halt germination and nuclear division in the presence of hydroxyurea than wild type, suggesting checkpoint defects. ATRIP proteins bind tightly to ATR PI-3 kinase (phosphatidylinositol 3-kinase) proteins. Therefore, we searched the Neurospora genome sequence for homologues of the Aspergillus nidulans ATR, UvsB. A uvsB homologous sequence was present in the right arm of chromosome I where the mus-9 gene maps. A cosmid containing this genomic DNA complemented the mus-9 mutation. The putative MUS-9 protein is 2484 amino acids long with eight introns. Homology is especially high in the C-terminal 350 amino acids that correspond to the PI-3 kinase domain. In wild type a low level of constitutive mRNA is present for both genes. It is transiently induced upon UV exposure.


Assuntos
Proteínas de Ciclo Celular/genética , Reparo do DNA , Proteínas de Ligação a DNA/genética , Epistasia Genética , Proteínas Fúngicas/genética , Neurospora crassa/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Primers do DNA/genética , Cinética , Metanossulfonato de Metila , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Mutação Puntual/genética , Polimorfismo de Fragmento de Restrição , Alinhamento de Sequência , Análise de Sequência de DNA , Raios Ultravioleta
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