RESUMO
In diploid eukaryotes, repair of double-stranded DNA breaks by homologous recombination often leads to loss of heterozygosity (LOH). Most previous studies of mitotic recombination in Saccharomyces cerevisiae have focused on a single chromosome or a single region of one chromosome at which LOH events can be selected. In this study, we used two techniques (single-nucleotide polymorphism microarrays and high-throughput DNA sequencing) to examine genome-wide LOH in a diploid yeast strain at a resolution averaging 1 kb. We examined both selected LOH events on chromosome V and unselected events throughout the genome in untreated cells and in cells treated with either γ-radiation or ultraviolet (UV) radiation. Our analysis shows the following: (1) spontaneous and damage-induced mitotic gene conversion tracts are more than three times larger than meiotic conversion tracts, and conversion tracts associated with crossovers are usually longer and more complex than those unassociated with crossovers; (2) most of the crossovers and conversions reflect the repair of two sister chromatids broken at the same position; and (3) both UV and γ-radiation efficiently induce LOH at doses of radiation that cause no significant loss of viability. Using high-throughput DNA sequencing, we also detected new mutations induced by γ-rays and UV. To our knowledge, our study represents the first high-resolution genome-wide analysis of DNA damage-induced LOH events performed in any eukaryote.
Assuntos
Raios gama , Genoma Fúngico , Perda de Heterozigosidade , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Raios Ultravioleta , Cromátides/genética , Cromátides/efeitos da radiação , Mapeamento Cromossômico , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/efeitos da radiação , Troca Genética , Dano ao DNA , DNA Fúngico/genética , Diploide , Sequenciamento de Nucleotídeos em Larga Escala , Meiose , Mitose , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
We employed a genetic assay based on illegitimate hybridization of heterothallic Saccharomyces cerevisiae strains (the α-test) to analyze the consequences for genome stability of inactivating translesion synthesis (TLS) DNA polymerases. The α-test is the only assay that measures the frequency of different types of mutational changes (point mutations, recombination, chromosome or chromosome arm loss) and temporary changes in genetic material simultaneously. All these events are manifested as illegitimate hybridization and can be distinguished by genetic analysis of the hybrids and cytoductants. We studied the effect of Polζ, Polη, and Rev1 deficiency on the genome stability in the absence of genotoxic treatment and in UV-irradiated cells. We show that, in spite of the increased percent of accurately repaired primary lesions, chromosome fragility, rearrangements, and loss occur in the absence of Polζ and Polη. Our findings contribute to further refinement of the current models of translesion synthesis and the organization of eukaryotic replication fork.
Assuntos
Cromossomos Fúngicos/genética , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Cromossomos Fúngicos/metabolismo , Cromossomos Fúngicos/efeitos da radiação , DNA Polimerase Dirigida por DNA/genética , Instabilidade Genômica/efeitos da radiação , Mutação/efeitos da radiação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/genética , Raios UltravioletaRESUMO
Ultraviolet (UV) light induces DNA-damage checkpoints and mutagenesis, which are involved in cancer protection and tumorigenesis, respectively. How cells identify DNA lesions and convert them to checkpoint-activating structures is a major question. We show that during repair of UV lesions in noncycling cells, Exo1-mediated processing of nucleotide excision repair (NER) intermediates competes with repair DNA synthesis. Impediments of the refilling reaction allow Exo1 to generate extended ssDNA gaps, detectable by electron microscopy, which drive Mec1 kinase activation and will be refilled by long-patch repair synthesis, as shown by DNA combing. We provide evidence that this mechanism may be stimulated by closely opposing UV lesions, represents a strategy to redirect problematic repair intermediates to alternative repair pathways, and may also be extended to physically different DNA damages. Our work has significant implications for understanding the coordination between repair of DNA lesions and checkpoint pathways to preserve genome stability.
Assuntos
Ciclo Celular , Cromossomos Fúngicos , Dano ao DNA , Reparo do DNA , DNA Fúngico/metabolismo , DNA de Cadeia Simples/metabolismo , Exodesoxirribonucleases/metabolismo , Saccharomyces cerevisiae/enzimologia , Ciclo Celular/genética , Ciclo Celular/efeitos da radiação , Cromossomos Fúngicos/efeitos da radiação , Cromossomos Fúngicos/ultraestrutura , Reparo do DNA/efeitos da radiação , DNA Fúngico/efeitos da radiação , DNA Fúngico/ultraestrutura , DNA de Cadeia Simples/ultraestrutura , Relação Dose-Resposta à Radiação , Ativação Enzimática , Exodesoxirribonucleases/genética , Regulação Fúngica da Expressão Gênica , Instabilidade Genômica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Tempo , Raios UltravioletaRESUMO
Radiobiological effects of homozygous Saccharomyces cerevisae strains of different ploidy from haploid to hexaploid were studied. Radiation (gamma-radiation of 60Co and fast 0.85 MeV neutrons) inactivation showed the minimum of resistance of haploid strain, the maximum of resistance of diploid strain and a decrease of resistance with further increase genome ploidy. All studied strains (except haploid) have the same capability to recovery in non-nutrient media during incubation at postradiation period by gamma-radiation and fast neutrons damage. Also it was found that values relative biological effectiveness (RBE) of fast neutrons for lethality are 2.6-2.7 and is independents from ploidy (2 and higher).
Assuntos
Nêutrons Rápidos , Raios gama , Saccharomyces cerevisiae/efeitos da radiação , Cromossomos Fúngicos/efeitos da radiação , Haploidia , Ploidias , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
The structural maintenance of chromosome (SMC) proteins constitute the cores of three protein complexes involved in chromosome metabolism; cohesin, condensin and the Smc5-Smc6 complex. While the roles of cohesin and condensin in sister chromatid cohesion and chromosome condensation respectively have been described, the cellular function of Smc5-Smc6 is as yet not understood, consequently the less descriptive name. The complex is involved in a variety of DNA repair pathways. It contains activities reminiscent of those described for cohesin and condensin, as well as several DNA helicases and endonucleases. It is required for sister chromatid recombination, and smc5-smc6 mutants suffer from the accumulation of unscheduled recombination intermediates. The complex contains a SUMO-ligase and potentially an ubiquitin-ligase; thus Smc5-Smc6 might presently have a dull name, but it seems destined to be recognized as a key player in the maintenance of chromosome stability. In this review we summarize our present understanding of this enigmatic protein complex.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Cromossomos/fisiologia , Complexos Multiproteicos/fisiologia , Adenosina Trifosfatases/fisiologia , Animais , Cromátides/fisiologia , Cromátides/ultraestrutura , Proteínas Cromossômicas não Histona/fisiologia , Cromossomos/ultraestrutura , Cromossomos Fúngicos/efeitos dos fármacos , Cromossomos Fúngicos/fisiologia , Cromossomos Fúngicos/efeitos da radiação , Cromossomos Fúngicos/ultraestrutura , Reparo do DNA/fisiologia , Replicação do DNA/fisiologia , DNA Fúngico/genética , DNA Ribossômico/genética , Proteínas de Ligação a DNA/fisiologia , Humanos , Recombinação Genética/fisiologia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Schizosaccharomyces/citologia , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/genética , Schizosaccharomyces/efeitos da radiação , Proteínas de Schizosaccharomyces pombe/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Proteínas de Xenopus/fisiologia , Xenopus laevis/genética , CoesinasRESUMO
No neutron dose rate effects in the wide range of 10(-3) Gy/s to 10(6) Gy/s were observed in yeast diploid cells for induction of mitotic segregation and crossing-over. The RBE values for these effects were determined as doses ratio (Dgamma/D(n)) at maximum effects. The RBE were 2.2-1.9 for neutrons of the reactor BR-10 (E = = 0.85 MeV) and the pulse reactor BARS-6 (E = 1.44 MeV). The RBE values for genetic effects were 1.0 at the equal survival level for neutrons and gamma-rays 60Co.
Assuntos
Nêutrons , Saccharomyces cerevisiae/efeitos da radiação , Segregação de Cromossomos/efeitos da radiação , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/efeitos da radiação , Troca Genética/efeitos da radiação , Diploide , Relação Dose-Resposta à Radiação , Mutação , Saccharomyces cerevisiae/genéticaRESUMO
MEC1, the essential yeast ATM/ATR homolog, prevents replication fork collapse and is required for the cellular response to DNA damage. We had previously observed higher rates of spontaneous SCE, heteroallelic recombination and translocations in mec1-21 mutants, which still retain some G2 checkpoint function, compared to mec1 null mutants, which are completely defective in checkpoint function, and wild type. However, the types of DNA lesions that are more recombinogenic in mec1-21, compared to wild type, are unknown. Here, we measured DNA damage-associated SCE, homolog (heteroallelic) recombination, and homology-directed translocations in mec1-21, and characterized types of DNA damage-associated chromosomal rearrangements that occur in mec1-21. Although frequencies of UV-associated recombination were higher in mec1-21, the mutant was defective in double-strand break-associated SCE and heteroallelic recombination. Over-expression of Rad53 in mec1-21 reduced UV-associated recombination but did not suppress the defect in X-ray-associated recombination. Both X ray and UV exposure increased translocation frequencies in mec1-21, but the majority of the UV-associated products were non-reciprocal translocations. We suggest that although recombinational repair of double-stand breaks is less efficient in mec1 mutants, recombinants may be generated by other mechanisms, such as break-induced replication.
Assuntos
Recombinação Genética/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Troca de Cromátide Irmã/efeitos da radiação , Raios Ultravioleta , Raios X , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2 , Cromossomos Fúngicos/efeitos da radiação , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Peptídeos e Proteínas de Sinalização Intracelular , Modelos Biológicos , Organismos Geneticamente Modificados , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência , Translocação Genética/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Regulação para Cima/efeitos da radiação , Raios X/efeitos adversosRESUMO
Homologous recombination (HR) is a source of genomic instability and the loss of heterozygosity in mitotic cells. Since these events pose a severe health risk, it is important to understand the molecular events that cause spontaneous HR. In eukaryotes, high levels of HR are a normal feature of meiosis and result from the induction of a large number of DNA double-strand breaks (DSBs). By analogy, it is generally believed that the rare spontaneous mitotic HR events are due to repair of DNA DSBs that accidentally occur during mitotic growth. Here we provide the first direct evidence that most spontaneous mitotic HR in Saccharomyces cerevisiae is initiated by DNA lesions other than DSBs. Specifically, we describe a class of rad52 mutants that are fully proficient in inter- and intra-chromosomal mitotic HR, yet at the same time fail to repair DNA DSBs. The conclusions are drawn from genetic analyses, evaluation of the consequences of DSB repair failure at the DNA level, and examination of the cellular re-localization of Rad51 and mutant Rad52 proteins after introduction of specific DSBs. In further support of our conclusions, we show that, as in wild-type strains, UV-irradiation induces HR in these rad52 mutants, supporting the view that DNA nicks and single-stranded gaps, rather than DSBs, are major sources of spontaneous HR in mitotic yeast cells.
Assuntos
Quebras de DNA de Cadeia Dupla , Recombinação Genética/genética , Saccharomyces cerevisiae/genética , Alelos , Camptotecina/farmacologia , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , DNA Topoisomerases/metabolismo , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Raios gama , Cinética , Testes de Sensibilidade Microbiana , Mitose/efeitos dos fármacos , Mitose/fisiologia , Proteínas Mutantes/metabolismo , Mutação/genética , Fenótipo , Transporte Proteico/efeitos dos fármacos , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinação Genética/efeitos da radiação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/metabolismo , Raios UltravioletaRESUMO
Cells maintain genomic stability by the coordination of DNA-damage repair and cell-cycle checkpoint control. In replicating cells, DNA damage usually activates intra-S-phase checkpoint controls, which are characterized by delayed S-phase progression and increased Rad53 phosphorylation. We show that in budding yeast, the intra-S-phase checkpoint controls, although functional, are not activated by the topoisomerase I inhibitor camptothecin (CPT). In a CPT-hypersensitive mutant strain that lacks the histone 2A (H2A) phosphatidylinositol-3-OH kinase (PI(3)K) motif at Ser 129 (h2a-s129a), the hypersensitivity was found to result from a failure to process full-length chromosomal DNA molecules during ongoing replication. H2A Ser 129 is not epistatic to the RAD24 and RAD9 checkpoint genes, suggesting a non-checkpoint role for the H2A PI(3)K site. These results suggest that H2A Ser 129 is an essential component for the efficient repair of DNA double-stranded breaks (DSBs) during replication in yeast, particularly of those DSBs that do not induce the intra-S-phase checkpoint.
Assuntos
Dano ao DNA/genética , Reparo do DNA/fisiologia , Histonas/química , Histonas/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Serina/metabolismo , Animais , Camptotecina/farmacologia , Ciclo Celular/fisiologia , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , Cromossomos Fúngicos/efeitos da radiação , Dano ao DNA/efeitos da radiação , DNA Topoisomerases Tipo I/metabolismo , Eletroforese em Gel de Campo Pulsado , Histonas/genética , Humanos , Mutação de Sentido Incorreto/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Serina/genética , Inibidores da Topoisomerase IRESUMO
Induction of DSBs in the diploid yeast, Saccharomyces cerevisiae, was measured by pulsed-field gel electrophoresis (PFGE) after the cells had been exposed on membrane filters to a variety of energetic heavy ions with values of linear energy transfer (LET) ranging from about 2 to 11,500 keV/microm, (241)Am alpha particles, and 80 keV X rays. After irradiation, the cells were lysed, and the chromosomes were separated by PFGE. The gels were stained with ethidium bromide, placed on a UV transilluminator, and analyzed using a computer-coupled camera. The fluorescence intensities of the larger bands were found to decrease exponentially with dose or particle fluence. The slope of this line corresponds to the cross section for at least one double-strand break (DSB), but closely spaced multiple breaks cannot be discriminated. Based on the known size of the native DNA molecules, breakage cross sections per base pair were calculated. They increased with LET until they reached a transient plateau value of about 6 x 10(-7) microm(2) at about 300-2000 keV/microm; they then rose for the higher LETs, probably reflecting the influence of delta electrons. The relative biological effectiveness for DNA breakage displays a maximum of about 2.5 around 100-200 keV/microm and falls below unity for LET values above 10(3) keV/microm. For these yeast cells, comparison of the derived breakage cross sections with the corresponding cross section for inactivation derived from the terminal slope of the survival curves shows a strong linear relationship between these cross sections, extending over several orders of magnitude.
Assuntos
Cromossomos Fúngicos/efeitos da radiação , Dano ao DNA/efeitos da radiação , DNA Fúngico/efeitos da radiação , Íons Pesados/efeitos adversos , Saccharomyces cerevisiae/efeitos da radiação , Partículas alfa , Quebra Cromossômica , Relação Dose-Resposta à Radiação , Eletroforese em Gel de Campo Pulsado , Transferência Linear de Energia , Prótons , Eficiência Biológica Relativa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Raios XRESUMO
Chromosomes of budding yeast Saccharomyces pastorianus were used to determine the extent of DNA double-strand breaks (DSBs) induced by x-rays (30-50 keV) and 14 MeV neutrons. The yeast chromosomes were separated by pulsed-field gel electrophoresis (PFGE) and the proportion of unbroken molecules corresponding to the largest chromosome no. IV (1500 kbp) was used to calculate the DSB frequency assuming a random distribution of hits. To determine the protective contribution of the cell environment, chromosomes embedded in agarose plugs as well as intact yeast cells, were irradiated under conditions completely inhibiting DNA repair. Following irradiation, the intact cells were also embedded in agarose plugs and the chromosomes isolated to perform PFGE. All radiation experiments resulted in a linear dose-effect curve for DSBs. For both radiation qualities, the yield of DSBs for exposed isolated chromosomes exceeded that for intact yeast cells by a factor of 13. The relative biological effectiveness (RBE) of 14 MeV neutrons in the induction of DNA DSBs was about 2.5. This figure was found to be identical for the in vivo and in vitro exposure of yeast chromosomes (neutrons 36.7 and 2.8, x-rays 14.5 and 1.1 x 10(-8) DSB x Bp-1 Gy-1 for isolated DNA and intact cells, respectively).
Assuntos
Cromossomos Fúngicos/efeitos da radiação , Dano ao DNA , DNA Fúngico/efeitos da radiação , Saccharomyces/efeitos da radiação , DNA Fúngico/isolamento & purificação , Relação Dose-Resposta à Radiação , Eletroforese em Gel de Campo Pulsado/métodos , Nêutrons , Saccharomyces/genética , Sefarose , Raios XRESUMO
In this study a simple electrophoresis approach has been proposed for assessing DNA damage per chromosome in vitro. Novel procedures of gel casting, sample loading, electrophoresis and quantification of damage have been suggested. Sets of Saccharomyces cerevisiae chromosomes subjected to DNA damage by Bleomycin, Co60-gamma-radiation alone and in combination with Hoechst were studied in detail. Statistical analyses showed that damage induced by Bleomycin bore linear positive correlation with %GA (r = 0.97) and %GT (r = 0.61) contents of chromosomes. Samples pre-treated with Hoechst showed much less damage by Co60-gamma-irradiation as compared to samples not treated with Hoechst but exposed to Co60-gamma-irradiation. The 'protective effect of Hoechst' bore linear positive correlation (r = 0.8) with %TAT content of chromosomes.
Assuntos
Cromossomos Fúngicos/efeitos dos fármacos , Cromossomos Fúngicos/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Eletroforese em Gel de Ágar/métodos , Bleomicina/farmacologia , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , DNA Fúngico/química , DNA Fúngico/efeitos dos fármacos , DNA Fúngico/metabolismo , DNA Fúngico/efeitos da radiação , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Eletroforese em Gel Bidimensional , Raios gama , Saccharomyces cerevisiae/genéticaRESUMO
Poly(dA.dT) sequences (T-tracts) are abundant genomic DNA elements with unusual properties in vitro and an established role in transcriptional regulation of yeast genes. In vitro T-tracts are rigid, contribute to DNA bending, affect assembly in nucleosomes and generate a characteristic pattern of CPDs (cyclobutane pyrimidine dimers) upon irradiation with UV light (UV photofootprint). In eukaryotic cells, where DNA is packaged in chromatin, the DNA structure of T-tracts is unknown. Here we have used in vivo UV photofootprinting and DNA repair by photolyase to investigate the structure and accessibility of T-tracts in yeast promoters (HIS3, URA3 and ILV1). The same characteristic photofootprints were obtained in yeast and in naked DNA, demonstrating that the unusual T-tract structure exists in living cells. Rapid repair of CPDs in the T-tracts demonstrates that these T-tracts were not folded in nucleosomes. Moreover, neither datin, a T-tract binding protein, nor Gcn5p, a histone acetyltransferase involved in nucleosome remodelling, showed an influence on the structure and accessibility of T-tracts. The data support a contribution of this unusual DNA structure to transcriptional regulation.
Assuntos
DNA Fúngico/química , DNA Fúngico/genética , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Conformação de Ácido Nucleico , Nucleossomos/fisiologia , Poli A/genética , Poli T/genética , Regiões Promotoras Genéticas/genética , Proteínas de Saccharomyces cerevisiae , Leveduras/genética , Acetiltransferases/genética , Acetiltransferases/metabolismo , Sequência de Bases , Cromossomos Fúngicos/química , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , Cromossomos Fúngicos/efeitos da radiação , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , Pegada de DNA , Reparo do DNA/genética , DNA Fúngico/metabolismo , DNA Fúngico/efeitos da radiação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desoxirribodipirimidina Fotoliase/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Genoma Fúngico , Histona Acetiltransferases , Hidroliases/genética , Maleabilidade , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Raios Ultravioleta , Leveduras/enzimologia , Leveduras/efeitos da radiaçãoRESUMO
Primary gene amplification, the mutation from one gene copy per genome to two or more copies per genome, is a major mechanism of oncogene overexpression in human cancers. Analysis of the structures of amplifications can provide important evidence about the mechanism of amplification formation. We report here the analysis of the structures of four independent spontaneous circular amplifications of ADH4:CUP1 in the yeast Saccharomyces cerevisiae. The structures of all four amplifications are consistent with their formation by a breakage-fusion-bridge (BFB) mechanism. All four of these amplifications include a centromere as predicted by the BFB model. All four of the amplifications have a novel joint located between the amplified DNA and the telomere, which results in a dicentric chromosome, and is adjacent to all the copies of the amplified DNA as predicted by the BFB model. In addition we demonstrated that two of the amplifications contain most of chromosome VII in an unrearranged form in a 1:1 ratio with the normal copy of chromosome VII, again consistent with the predictions of the BFB model. Finally, all four amplifications are circular, one stable endpoint for molecules after breakage- fusion-bridge.
Assuntos
Amplificação de Genes , Saccharomyces cerevisiae/genética , Proteínas de Transporte , Centrômero/genética , Cromossomos Fúngicos/efeitos da radiação , DNA Circular , Raios gama , Dosagem de Genes , Vetores Genéticos/genética , Metalotioneína/genética , Modelos Genéticos , Sequências Repetitivas de Ácido Nucleico , Saccharomyces cerevisiae/efeitos da radiação , Telômero/genéticaRESUMO
Effects of Modifying Topoisomerase II Levels on Cellular Recovery from Radiation Damage. Experiments were performed with the budding yeast, Saccharomyces cerevisiae, to test whether DNA topoisomerase II is involved in repair of DNA damage induced by ionizing radiation. Topoisomerase II was inactivated by use of a temperature-sensitive mutation. Enzyme inactivation increased cellular radiosensitivity, blocked the restitution of broken chromosomes, assayed by pulsed-field gel electrophoresis, and prolonged the induction of a DNA damage-inducible gene (RNR3). Overexpression of the topoisomerase II gene did not alter cellular radiosensitivity. The data support a role for topoisomerase II in the repair of DNA strand breaks.
Assuntos
Dano ao DNA , Reparo do DNA , DNA Topoisomerases Tipo II/fisiologia , DNA Fúngico/efeitos da radiação , Proteínas Fúngicas/fisiologia , Saccharomyces cerevisiae/efeitos da radiação , Cromossomos Fúngicos/efeitos da radiação , Cromossomos Fúngicos/ultraestrutura , DNA Topoisomerases Tipo II/genética , DNA Fúngico/genética , DNA Fúngico/metabolismo , Eletroforese em Gel de Campo Pulsado , Indução Enzimática , Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Genes Reporter , Temperatura Alta , Novobiocina/farmacologia , Regiões Promotoras Genéticas , Tolerância a Radiação/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Inibidores da Topoisomerase IIRESUMO
The checkpoint mechanisms that delay cell cycle progression in response to DNA damage or inhibition of DNA replication are necessary for maintenance of genetic stability in eukaryotic cells. Potential targets of checkpoint-mediated regulation include proteins directly involved in DNA metabolism, such as the cellular single-stranded DNA (ssDNA) binding protein, replication protein A (RPA). Studies in Saccharomyces cerevisiae have revealed that the RPA large subunit (Rfa1p) is involved in the G1 and S phase DNA damage checkpoints. We now demonstrate that Rfa1p is phosphorylated in response to various forms of genotoxic stress, including radiation and hydroxyurea exposure, and further show that phosphorylation of Rfa1p is dependent on the central checkpoint regulator Mec1p. Analysis of the requirement for other checkpoint genes indicates that different mechanisms mediate radiation- and hydroxyurea-induced Rfa1p phosphorylation despite the common requirement for functional Mec1p. In addition, experiments with mutants defective in the Cdc13p telomere-binding protein indicate that ssDNA formation is an important signal for Rfa1p phosphorylation. Because Rfa1p contains the major ssDNA binding activity of the RPA heterotrimer and is required for DNA replication, repair and recombination, it is possible that phosphorylation of this subunit is directly involved in modulating RPA activity during the checkpoint response.
Assuntos
Ciclo Celular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Fatores de Transcrição , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Cromossomos Fúngicos/efeitos dos fármacos , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , Cromossomos Fúngicos/efeitos da radiação , Ciclina B/genética , Ciclina B/metabolismo , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , Reparo do DNA , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/efeitos da radiação , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteína Quinase Ativada por DNA , Relação Dose-Resposta à Radiação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos/genética , Glicosiltransferases/metabolismo , Humanos , Hidroxiureia/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Mutação/genética , Proteínas Nucleares , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Proteínas Serina-Treonina Quinases/metabolismo , Proteína de Replicação A , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Telômero/efeitos dos fármacos , Telômero/genética , Telômero/metabolismo , Telômero/efeitos da radiação , Raios UltravioletaRESUMO
Spo11, a type II topoisomerase, is likely to be required universally for initiation of meiotic recombination. However, a dichotomy exists between budding yeast and the animals Caenorhabditis elegans and Drosophila melanogaster with respect to additional roles of Spo11 in meiosis. In Saccharomyces cerevisiae, Spo11 is required for homolog pairing, as well as axial element (AE) and synaptonemal complex (SC) formation. All of these functions are Spo11 independent in C.elegans and D.melanogaster. We examined Spo11 function in a multicellular fungus, Coprinus cinereus. The C.cinereus spo11-1 mutant shows high levels of homolog pairing and occasionally forms full-length AEs, but no SC. In C.cinereus, Spo11 is also required for maintenance of meiotic chromosome condensation and proper spindle formation. Meiotic progression in spo11-1 is aberrant; late in meiosis basidia undergo programmed cell death (PCD). To our knowledge, this is the first example of meiotic PCD outside the animal kingdom. Ionizing radiation can partially rescue spo11-1 for both AE and SC formation and viable spore production, suggesting that the double-strand break function of Spo11 is conserved and is required for these functions.
Assuntos
Cromossomos Fúngicos/fisiologia , Coprinus/enzimologia , DNA Topoisomerases Tipo II/fisiologia , Esterases/fisiologia , Proteínas Fúngicas/fisiologia , Meiose/fisiologia , Sequência de Aminoácidos , Apoptose , Cromossomos Fúngicos/efeitos da radiação , Coprinus/citologia , Coprinus/genética , Coprinus/efeitos da radiação , DNA Topoisomerases Tipo II/genética , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA Fúngico/efeitos da radiação , Endodesoxirribonucleases , Esterases/genética , Proteínas Fúngicas/genética , Dados de Sequência Molecular , Prófase , Alinhamento de Sequência , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Fuso Acromático/fisiologia , Fuso Acromático/ultraestrutura , Complexo SinaptonêmicoRESUMO
Repair of UV-induced cyclobutane pyrimidine dimers (CPDs) was measured in a yeast minichromosome, having a galactose-inducible GAL1:URA3 fusion gene, a constitutively expressed HIS3 gene and varied regions of chromatin structure. Transcription of GAL1:URA3 increased >150-fold, while HIS3 expression decreased <2-fold when cells were switched from glucose to galactose medium. Following galactose induction, four nucleosomes were displaced or rearranged in the GAL3-GAL10 region. However, no change in nucleosome arrangement was observed in other regions of the minichromosome following induction, indicating that only a few plasmid molecules actively transcribe at any one time. Repair at 269 cis-syn CPD sites revealed moderate preferential repair of the transcribed strand of GAL1:URA3 in galactose, consistent with transcription-coupled repair in a fraction of these genes. Many sites upstream of the transcription start site in the transcribed strand were also repaired faster upon induction. There is remarkable repair heterogeneity in the HIS3 gene and preferential repair is seen only in a short sequence immediately downstream of the transcription start site. Finally, a mild correlation of repair heterogeneity with nucleosome positions was observed in the transcribed strand of the inactive GAL1:URA3 gene and this correlation was abolished upon galactose induction.
Assuntos
Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , Reparo do DNA , Dímeros de Pirimidina/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cromatina/metabolismo , Cromossomos Fúngicos/efeitos da radiação , DNA Fúngico/análise , DNA Fúngico/efeitos da radiação , Genes Reporter/genética , Modelos Genéticos , Plasmídeos , RNA Fúngico/análise , RNA Fúngico/efeitos da radiação , Fatores de Tempo , Transcrição Gênica , Raios UltravioletaRESUMO
Radiation-induced chromosome aberrations, particularly exchange-type aberrations, are thought to result from misrepair of DNA double-strand breaks. The relationship between individual pathways of break repair and aberration formation is not clear. By electrophoretic karyotyping of single-cell clones derived from irradiated cells, we have analyzed the induction of stable aberrations in haploid yeast cells mutated for the RAD52 gene, the RAD54 gene, the HDF1(= YKU70) gene, or combinations thereof. We found low and comparable frequencies of aberrational events in wildtype and hdf1 mutants, and assume that in these strains most of the survivors descended from cells that were in G2 phase during irradiation and therefore able to repair breaks by homologous recombination between sister chromatids. In the rad52 and the rad54 strains, enhanced formation of aberrations, mostly exchange-type aberrations, was detected, demonstrating the misrepair activity of a rejoining mechanism other than homologous recombination. No aberration was found in the rad52 hdf1 double mutant, and the frequency in the rad54 hdf1 mutant was very low. Hence, misrepair resulting in exchange-type aberrations depends largely on the presence of Hdf1, a component of the nonhomologous end-joining pathway in yeast.
Assuntos
Aberrações Cromossômicas , Cromossomos Fúngicos/efeitos da radiação , Reparo do DNA , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/efeitos da radiação , Cromossomos Fúngicos/genética , DNA Helicases , Enzimas Reparadoras do DNA , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Raios gama , Cariotipagem , Proteína Rad52 de Recombinação e Reparo de DNA , Saccharomyces cerevisiae/genéticaRESUMO
Electrophoretic karyotypes of 80 auxotrophic and morphological mutants obtained from two Phaffia rhodozyma strains (ATCC 24203 and ATCC 24229) by gamma-radiation were investigated. Contour-clamped homogeneous gel electrophoresis separation of the chromosomal size DNAs revealed 29 new chromosomal patterns after mutagen treatment. No correlation was found between a given type of chromosomal aberration and any phenotypic character. However, analysis of the chromosomal rearrangements proved to be useful for a more exact determination of chromosome number and genome size. The total genome size of ATCC 24229 was found to be 19.3 Mb, with nine chromosomes, while analysis of the mutant derivatives of ATCC 24203 suggested the presence of 11 chromosomes, with an estimated total genome size of 22.2 Mb. The advantages of the analysis of mutant electrophoretic karyotypes for genome characterization are discussed.