RESUMO
The Cdc7-Dbf4 complex is a conserved serine/threonine protein kinase essential for the initiation of eukaryotic DNA replication. Although an mcm5-bob1 mutation bypasses lethality conferred by mutations in CDC7 or DBF4, the Deltacdc7 mcm5-bob1 mutant is sensitive to hydroxyurea (HU), which induces replication stress. To elucidate the reasons for HU sensitivity conferred by deletion of CDC7, we examined the role of Cdc7-Dbf4 in the replication checkpoint. We found that in Cdc7-Dbf4-deficient cells exposed to replication stress, Rad53 remains in a hypophosphorylated form, anaphase spindle is elongated, and checkpoint-specific transcription is not induced. The hypophosphorylated Rad53 exhibits a low autophosphorylation activity, and recombinant Cdc7-Dbf4 phosphorylates Rad53 in vitro. These results suggest that Cdc7-Dbf4 is required for full activation of Rad53 in response to replication stress.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Replicação do DNA , DNA Fúngico/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Anáfase , Western Blotting , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2 , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Hidroxiureia/farmacologia , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Fase S/fisiologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , beta-Galactosidase/metabolismoRESUMO
Meiosis ensures genetic diversification of gametes and sexual reproduction. For successful meiosis, multiple events such as DNA replication, recombination, and chromosome segregation must occur coordinately in a strict regulated order. We investigated the meiotic roles of Cdc7 kinase in the initiation of meiotic recombination, namely, DNA double-strand breaks (DSBs) mediated by Spo11 and other coactivating proteins. Genetic analysis using bob1-1 cdc7Delta reveals that Cdc7 is essential for meiotic DSBs and meiosis I progression. We also demonstrate that the N-terminal region of Mer2, a Spo11 ancillary protein required for DSB formation and phosphorylated by cyclin-dependent kinase (CDK), contains two types of Cdc7-dependent phosphorylation sites near the CDK site (Ser30): One (Ser29) is essential for meiotic DSB formation, and the others exhibit a cumulative effect to facilitate DSB formation. Importantly, mutations on these sites confer severe defects in DSB formation even when the CDK phosphorylation is present at Ser30. Diploids of cdc7Delta display defects in the chromatin binding of not only Spo11 but also Rec114 and Mei4, other meiotic coactivators that may assist Spo11 binding to DSB hot spots. We thus propose that Cdc7, in concert with CDK, regulates Spo11 loading to DSB sites via Mer2 phosphorylation.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Quebras de DNA de Cadeia Dupla , Meiose/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/fisiologia , Sequência de Aminoácidos , Replicação do DNA/fisiologia , DNA Topoisomerases Tipo II/metabolismo , Endodesoxirribonucleases , Dados de Sequência Molecular , Fosforilação , Recombinação Genética/genética , Recombinação Genética/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The assembly of the prereplicative complex (pre-RC) at the origin of replication in eukaryotes is a highly regulated and highly conserved process that plays a critical role in preventing multiple rounds of DNA replication per cell division cycle. This study analyzes the molecular dynamics of the assembly of Saccharomyces cerevisiae pre-RC in vitro using ARS1 plasmid DNA and yeast whole cell extracts. In addition, pre-RC assembly was reconstituted in vitro using ARS1 DNA and purified origin-recognition complex (ORC), Cdc6p and Cdt1p-Mcm2-7p. The results reveal sequential recruitment of ORC, Cdc6p, Cdt1p and Mcm2-7p on to ARS1 DNA. When Mcm2-7p is maximally loaded, Cdc6p and Cdt1p are released, suggesting that these two proteins are co-ordinately regulated during pre-RC assembly. In extracts from sid2-21 mutant cells that are deficient in CDT1, ORC and Cdc6p bind to ARS1 but Cdt1p and Mcm2-7p do not. However, Mcm2-7p does bind in the presence of exogenous Cdt1p or Cdt1p-Mcm2-7p complex. Cdt1p-Mcm2-7p complex, which was purified from G1-, early S or G2/M-arrested cells, exhibits structure-specific DNA binding, interacting only with bubble- or Y-shape-DNA, but the biological significance of this observation is not yet known.
Assuntos
Replicação do DNA/fisiologia , Origem de Replicação/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Proteínas de Ciclo Celular/genética , Divisão Celular/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Fase G1/genética , Fase G2/genética , Espectrometria de Massas , Complexo de Reconhecimento de Origem/genética , Fase S/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genéticaRESUMO
This manuscript was withdrawn at the request of the authors.
RESUMO
BACKGROUND: MCM10 is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae. Mcm10p functionally interacts with components of the pre-replicative complex (Mcm2-Mcm7 complex and origin recognition complex) as well as the pre-initiation complex component (Cdc45p) suggesting that it may be a component of the pre-RC as well as the pre-IC. Two-dimensional gel electrophoresis analysis showed that Mcm10p is required not only for the initiation of DNA synthesis at replication origins but also for the smooth passage of replication forks at origins. Genetic analysis showed that MCM10 interacts with components of the elongation machinery such as Pol delta and Pol epsilon, suggesting that it may play a role in elongation replication. RESULTS: We show that the mcm10 mutation causes replication fork pausing not only at potentially active origins but also at silent origins. We screened for mutations that are lethal in combination with mcm10-1 and obtained seven mutants named slm1-slm6 for synthetically lethal with mcm10. These mutants comprised six complementation groups that can be divided into three classes. Class 1 includes genes that encode components of the pre-RC and pre-IC and are represented by SLM3, 4 and 5 which are allelic to MCM7, MCM2 and CDC45, respectively. Class 2 includes genes involved in the processing of Okazaki fragments in lagging strand synthesis and is represented by SLM1, which is allelic to DNA2. Class 3 includes novel DNA repair genes represented by SLM2 and SLM6. CONCLUSIONS: The viability of the mcm10-1 mutant is dependent on a novel repair pathway that may participate either in resolving accumulated replication intermediates or the damage caused by blocked replication forks. These results are consistent with the hypothesis that Mcm10p is required for the passage of replication forks through obstacles such as those created by pre-RCs assembled at active or inactive replication origins.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Sobrevivência Celular , Reparo do DNA , Replicação do DNA , DNA Fúngico/metabolismo , Saccharomycetales/genética , Proteínas de Transporte , Proteínas de Ciclo Celular/genética , DNA , Proteínas de Ligação a DNA , Eletroforese em Gel Bidimensional , Proteínas Fúngicas , Genes Letais , Componente 7 do Complexo de Manutenção de Minicromossomo , Proteínas de Manutenção de Minicromossomo , Mutagênese Sítio-Dirigida , Mutação/genética , Proteínas Nucleares , Proteínas de Ligação a RNA , Origem de Replicação , Fase S , Proteínas de Saccharomyces cerevisiae , Saccharomycetales/metabolismo , Transdução de SinaisRESUMO
We report that POL5 encodes the fifth essential DNA polymerase in Saccharomyces cerevisiae. Pol5p was identified and purified from yeast cell extracts and is an aphidicolin-sensitive DNA polymerase that is stimulated by yeast proliferating cell nuclear antigen (PCNA). Thus, we named Pol5p DNA polymerase phi. Temperature-sensitive pol5-1-- -3 mutants did not arrest at G(2)/M at the restrictive temperature. Furthermore, the polymerase active-site mutant POL5dn gene complements the lethality of Delta pol5. These results suggest that the polymerase activity of Pol5p is not required for the in vivo function of Pol5p. rRNA synthesis was severely inhibited at the restrictive temperature in the temperature-sensitive pol5-3 mutant cells, suggesting that an essential function of Pol5p is rRNA synthesis. Pol5p is localized exclusively to the nucleolus and binds near or at the enhancer region of rRNA-encoding DNA repeating units.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Afidicolina/farmacologia , Sequência de Bases , Sítios de Ligação/genética , Divisão Celular , Nucléolo Celular/enzimologia , Replicação do DNA , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/isolamento & purificação , Elementos Facilitadores Genéticos , Genes Fúngicos , Dados de Sequência Molecular , Mutação , RNA Fúngico/biossíntese , RNA Fúngico/genética , RNA Ribossômico/biossíntese , RNA Ribossômico/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , TemperaturaRESUMO
Saccharomyces cerevisiae POL2 encodes the catalytic subunit of DNA polymerase epsilon. This study investigates the cellular functions performed by the polymerase domain of Pol2p and its role in DNA metabolism. The pol2-16 mutation has a deletion in the catalytic domain of DNA polymerase epsilon that eliminates its polymerase and exonuclease activities. It is a viable mutant, which displays temperature sensitivity for growth and a defect in elongation step of chromosomal DNA replication even at permissive temperatures. This mutation is synthetic lethal in combination with temperature-sensitive mutants or the 3'- to 5'-exonuclease-deficient mutant of DNA polymerase delta in a haploid cell. These results suggest that the catalytic activity of DNA polymerase epsilon participates in the same pathway as DNA polymerase delta, and this is consistent with the observation that DNA polymerases delta and epsilon colocalize in some punctate foci on yeast chromatids during S phase. The pol2-16 mutant senesces more rapidly than wild type strain and also has shorter telomeres. These results indicate that the DNA polymerase domain of Pol2p is required for rapid, efficient, and highly accurate chromosomal DNA replication in yeast.