RESUMO
Human p21(Waf1) protein is well known for being transcriptionally induced by p53 and activating the cell cycle checkpoint arrest in response to DNA breaks. Here we report that p21(Waf1) protein undergoes a bimodal regulation, being upregulated in response to low doses of DNA damage but rapidly and transiently degraded in response to high doses of DNA lesions. Responsible for this degradation is the checkpoint kinase Chk1, which phosphorylates p21(Waf1) on T145 and S146 residues and induces its proteasome-dependent proteolysis. The initial p21(Waf1) degradation is then counteracted by the ATM-Chk2 pathway, which promotes the p53-dependent accumulation of p21(Waf1) at any dose of damage. We also found that p21(Waf1) ablation favors the activation of an apoptotic program to eliminate otherwise irreparable cells. These findings support a model in which in human cells a balance between ATM-Chk2-p53 and the ATR-Chk1 pathways modulates p21(Waf1) protein levels in relation to cytostatic and cytotoxic doses of DNA damage.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Apoptose/efeitos dos fármacos , Bleomicina/farmacologia , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Regulação para Baixo/efeitos dos fármacos , Humanos , Fosforilação/efeitos dos fármacos , Proteínas Quinases/metabolismo , Proteólise/efeitos dos fármacosRESUMO
In response to DNA damage and blocks to replication, eukaryotes activate the checkpoint pathways that prevent genomic instability and cancer by coordinating cell cycle progression with DNA repair. In budding yeast, the checkpoint response requires the Mec1-dependent activation of the Rad53 protein kinase. Active Rad53 slows DNA synthesis when DNA is damaged and prevents firing of late origins of replication. Further, rad53 mutants are unable to recover from a replication block. Mec1 and Rad53 also modulate the phosphorylation state of different DNA replication and repair enzymes. Little is known of the mechanisms by which checkpoint pathways interact with the replication apparatus when DNA is damaged or replication blocked. We used the two-dimensional gel technique to examine replication intermediates in response to hydroxyurea-induced replication blocks. Here we show that hydroxyurea-treated rad53 mutants accumulate unusual DNA structures at replication forks. The persistence of these abnormal molecules during recovery from the hydroxyurea block correlates with the inability to dephosphorylate Rad53. Further, Rad53 is required to properly maintain stable replication forks during the block. We propose that Rad53 prevents collapse of the fork when replication pauses.
Assuntos
Proteínas de Ciclo Celular , Replicação do DNA , DNA Fúngico/biossíntese , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas de Saccharomyces cerevisiae , Ciclo Celular/genética , Ciclo Celular/fisiologia , Quinase do Ponto de Checagem 2 , DNA Fúngico/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Hidroxiureia/farmacologia , Mutação , Conformação de Ácido Nucleico , Inibidores da Síntese de Ácido Nucleico/farmacologia , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Origem de Replicação , Ribonucleotídeo Redutases/antagonistas & inibidores , SaccharomycetalesRESUMO
In Saccharomyces cerevisiae the rate of DNA replication is slowed down in response to DNA damage as a result of checkpoint activation, which is mediated by the Mec1 and Rad53 protein kinases. We found that the Srs2 DNA helicase, which is involved in DNA repair and recombination, is phosphorylated in response to intra-S DNA damage in a checkpoint-dependent manner. DNA damage-induced Srs2 phosphorylation also requires the activity of the cyclin-dependent kinase Cdk1, suggesting that the checkpoint pathway might modulate Cdk1 activity in response to DNA damage. Moreover, srs2 mutants fail to activate Rad53 properly and to slow down DNA replication in response to intra-S DNA damage. The residual Rad53 activity observed in srs2 cells depends upon the checkpoint proteins Rad17 and Rad24. Moreover, DNA damage-induced lethality in rad17 mutants depends partially upon Srs2, suggesting that a functional Srs2 helicase causes accumulation of lethal events in a checkpoint-defective context. Altogether, our data implicate Srs2 in the Mec1 and Rad53 pathway and connect the checkpoint response to DNA repair and recombination.
Assuntos
Proteína Quinase CDC2/metabolismo , DNA Helicases/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas de Saccharomyces cerevisiae , Western Blotting , Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular/metabolismo , Separação Celular , Quinase do Ponto de Checagem 2 , Dano ao DNA , DNA Helicases/genética , Reparo do DNA , Proteínas de Ligação a DNA , Citometria de Fluxo , Proteínas Fúngicas/genética , Genótipo , Peptídeos e Proteínas de Sinalização Intracelular , Metanossulfonato de Metila/farmacologia , Modelos Genéticos , Mutagênese Sítio-Dirigida , Proteínas Nucleares , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Plasmídeos/genética , Plasmídeos/metabolismo , Testes de Precipitina , Proteínas Quinases/metabolismo , Recombinação Genética , Fase S , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Temperatura , Fatores de TempoRESUMO
The Saccharomyces cerevisiae Rad53 protein kinase is required for the execution of checkpoint arrest at multiple stages of the cell cycle. We found that Rad53 autophosphorylation activity depends on in trans phosphorylation mediated by Mec1 and does not require physical association with other proteins. Uncoupling in trans phosphorylation from autophosphorylation using a rad53 kinase-defective mutant results in a dominant-negative checkpoint defect. Activation of Rad53 in response to DNA damage in G(1) requires the Rad9, Mec3, Ddc1, Rad17 and Rad24 checkpoint factors, while this dependence is greatly reduced in S phase cells. Furthermore, during recovery from checkpoint activation, Rad53 activity decreases through a process that does not require protein synthesis. We also found that Rad53 modulates the lagging strand replication apparatus by controlling phosphorylation of the DNA polymerase alpha-primase complex in response to intra-S DNA damage.
Assuntos
Proteínas de Ciclo Celular , Dano ao DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Ciclo Celular , Quinase do Ponto de Checagem 2 , Ativação Enzimática , Proteínas Fúngicas/metabolismo , Fase G1 , Genótipo , Modelos Genéticos , Mutagênese , Fosforilação , Fase S , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologiaRESUMO
Eukaryotic cells have evolved a network of control mechanisms, known as checkpoints, which coordinate cell-cycle progression in response to internal and external cues. The yeast Saccharomyces cerevisiae has been invaluable in dissecting genetically the DNA damage checkpoint pathway. Recent results on posttranslational modifications and protein-protein interactions of some key factors provide new insights into the architecture of checkpoint protein complexes and their order of function.
Assuntos
Dano ao DNA , Replicação do DNA , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Ciclo Celular/genética , Evolução Molecular , Genes Fúngicos , Modelos GenéticosRESUMO
Eukaryotic cells must be able to coordinate DNA repair, replication and cell cycle progression in response to DNA damage. A failure to activate the checkpoints which delay the cell cycle in response to internal and external cues and to repair the DNA lesions results in an increase in genetic instability and cancer predisposition. The use of the yeast Saccharomyces cerevisiae has been invaluable in isolating many of the genes required for the DNA damage response, although the molecular mechanisms which couple this regulatory pathway to different DNA transactions are still largely unknown. In analogy with prokaryotes, we propose that DNA strand breaks, caused by genotoxic agents or by replication-related lesions, trigger a replication coupled repair mechanism, dependent upon recombination, which is induced by the checkpoint acting during S-phase.
Assuntos
Dano ao DNA , Fase S , Schizosaccharomyces/genética , Schizosaccharomyces/citologiaRESUMO
Eukaryotic DNA replication is limited to once per cell cycle because cyclin-dependent kinases (cdks), which are required to fire origins, also prevent re-replication. Components of the replication apparatus, therefore, are 'reset' by cdk inactivation at the end of mitosis. In budding yeast, assembly of Cdc6p-dependent pre-replicative complexes (pre-RCs) at origins can only occur during G1 because it is blocked by cdk1 (Cdc28) together with B cyclins (Clbs). Here we describe a second, separate process which is also blocked by Cdc28/Clb kinase and, therefore, can only occur during G1; the recruitment of DNA polymerase alpha-primase (pol alpha) to chromatin. The recruitment of pol alpha to chromatin during G1 is independent of pre-RC formation since it can occur in the absence of Cdc6 protein. Paradoxically, overproduction of Cdc6p can drive both dephosphorylation and chromatin association of pol alpha. Overproduction of a mutant in which the N-terminus of Cdc6 has been deleted is unable to drive pol alpha chromatin binding. Since this mutant is still competent for pre-RC formation and DNA replication, we suggest that Cdc6p overproduction resets pol alpha chromatin binding by a mechanism which is independent of that used in pre-RC assembly.
Assuntos
Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/fisiologia , DNA Polimerase I/metabolismo , Replicação do DNA/fisiologia , Mitose/fisiologia , Proteínas de Saccharomyces cerevisiae , Cromatina/metabolismo , Proteínas Inibidoras de Quinase Dependente de Ciclina , DNA Primase/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiologia , Fase G1/fisiologia , Fosforilação , Proteínas Recombinantes de Fusão , Saccharomyces cerevisiae/genéticaRESUMO
Checkpoints prevent DNA replication or nuclear division when chromosomes are damaged. The Saccharomyces cerevisiae DDC1 gene belongs to the RAD17, MEC3 and RAD24 epistasis group which, together with RAD9, is proposed to act at the beginning of the DNA damage checkpoint pathway. Ddc1p is periodically phosphorylated during unperturbed cell cycle and hyperphosphorylated in response to DNA damage. We demonstrate that Ddc1p interacts physically in vivo with Mec3p, and this interaction requires Rad17p. We also show that phosphorylation of Ddc1p depends on the key checkpoint protein Mec1p and also on Rad24p, Rad17p and Mec3p. This suggests that Mec1p might act together with the Rad24 group of proteins at an early step of the DNA damage checkpoint response. On the other hand, Ddc1p phosphorylation is independent of Rad53p and Rad9p. Moreover, while Ddc1p is required for Rad53p phosphorylation, it does not play any major role in the phosphorylation of the anaphase inhibitor Pds1p, which requires RAD9 and MEC1. We suggest that Rad9p and Ddc1p might function in separated branches of the DNA damage checkpoint pathway, playing different roles in determining Mec1p activity and/or substrate specificity.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA/fisiologia , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , DNA Fúngico/genética , Proteínas Fúngicas/genética , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases , Saccharomyces cerevisiae/metabolismo , SecurinaRESUMO
The highly conserved DNA polymerase alpha-primase complex (pol-prism) is the only eukaryotic DNA polymerase that can initiate DNA synthesis de novo. It is required both for the initiation of DNA replication at chromosomal origins and for the discontinuous synthesis of Okazaki fragments on the lagging strand of the replication fork. The dual role of pol-prim makes it a likely target for mechanisms that control cell-cycle S-phase entry and progression.
Assuntos
Ciclo Celular , Dano ao DNA , DNA Primase/metabolismo , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Animais , HumanosRESUMO
The DDC1 gene was identified, together with MEC3 and other checkpoint genes, during a screening for mutations causing synthetic lethality when combined with a conditional allele altering DNA primase. Deletion of DDC1 causes sensitivity to UV radiation, methyl methanesulfonate (MMS) and hydroxyurea (HU). ddc1Delta mutants are defective in delaying G1-S and G2-M transition and in slowing down the rate of DNA synthesis when DNA is damaged during G1, G2 or S phase, respectively. Therefore, DDC1 is involved in all the known DNA damage checkpoints. Conversely, Ddc1p is not required for delaying entry into mitosis when DNA synthesis is inhibited. ddc1 and mec3 mutants belong to the same epistasis group, and DDC1 overexpression can partially suppress MMS and HU sensitivity of mec3Delta strains, as well as their checkpoint defects. Moreover, Ddc1p is phosphorylated periodically during a normal cell cycle and becomes hyperphosphorylated in response to DNA damage. Both phosphorylation events are at least partially dependent on a functional MEC3 gene.
Assuntos
Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Dano ao DNA , Genes Fúngicos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/metabolismo , Clonagem Molecular , Epistasia Genética , Glicoproteínas/farmacologia , Hidroxiureia/farmacologia , Metanossulfonato de Metila , Mutagênese , Mutagênicos/farmacologia , Periodicidade , Fosfoproteínas/metabolismo , Fosforilação , Supressão Genética , Raios Ultravioleta/efeitos adversosRESUMO
The temperature-sensitive yeast DNA primase mutant pri1-M4 fails to execute an early step of DNA replication and exhibits a dominant, allele-specific sensitivity to DNA-damaging agents. pri1-M4 is defective in slowing down the rate of S phase progression and partially delaying the G1-S transition in response to DNA damage. Conversely, the G2 DNA damage response and the S-M checkpoint coupling completion of DNA replication to mitosis are unaffected. The signal transduction pathway leading to Rad53p phosphorylation induced by DNA damage is proficient in pri1-M4, and cell cycle delay caused by Rad53p overexpression is counteracted by the pri1-M4 mutation. Altogether, our results suggest that DNA primase plays an essential role in a subset of the Rad53p-dependent checkpoint pathways controlling cell cycle progression in response to DNA damage.
Assuntos
Proteínas de Ciclo Celular , Dano ao DNA/genética , Replicação do DNA/genética , Proteínas Serina-Treonina Quinases , RNA Nucleotidiltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Western Blotting , Ciclo Celular/genética , Quinase do Ponto de Checagem 2 , DNA/biossíntese , DNA Primase , Estabilidade Enzimática/genética , Citometria de Fluxo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Genes Fúngicos/genética , Interfase/genética , Metanossulfonato de Metila/farmacologia , Mitose/genética , Modelos Biológicos , Mutagênese Sítio-Dirigida/genética , Mutagênicos/farmacologia , Mutação/genética , Fosforilação , Proteínas Quinases , RNA Nucleotidiltransferases/genética , Fase S/genética , Saccharomyces cerevisiae/genética , Temperatura , Raios Ultravioleta/efeitos adversosRESUMO
The rfa1-M2 and rfa1-M4 Saccharomyces cerevisiae mutants, which are altered in the 70 kDa subunit of replication protein A (RPA) and sensitive to UV and methyl methane sulfonate (MMS), have been analyzed for possible checkpoint defects. The G1/S and intra-S DNA damage checkpoints are defective in the rfa1-M2 mutant, since rfa1-M2 cells fail to properly delay cell cycle progression in response to UV irradiation in G1 and MMS treatment during S phase. Conversely, the G2/M DNA damage checkpoint and the S/M checkpoint are proficient in rfa1-M2 cells and all the checkpoints tested are functional in the rfa1-M4 mutant. Preventing S phase entry by alpha-factor treatment after UV irradiation in G1 does not change rfa1-M4 cell lethality, while it allows partial recovery of rfa1-M2 cell viability. Therefore, the hypersensitivity to UV and MMS treatments observed in the rfa1-M4 mutant might only be due to impairment of RPA function in DNA repair, while the rfa1-M2 mutation seems to affect both the DNA repair and checkpoint functions of Rpa70.
Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/química , Fase G1 , Fase S , Saccharomyces cerevisiae/genética , Células Cultivadas , Replicação do DNA , Proteínas de Ligação a DNA/fisiologia , Metanossulfonato de Metila/farmacologia , Peso Molecular , Mutagênese Sítio-Dirigida , Mutagênicos/farmacologia , Nocodazol/metabolismo , Proteína de Replicação A , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos da radiação , Relação Estrutura-Atividade , Raios UltravioletaRESUMO
The catalytic DNA primase subunit of the DNA polymerase alpha-primase complex is encoded by the essential PRI1 gene in Saccharomyces cerevisiae. To identify factors that functionally interact with yeast DNA primase in living cells, we developed a genetic screen for mutants that are lethal at the permissive temperature in a cold-sensitive pril-2 genetic background. Twenty-four recessive mutations belonging to seven complementation groups were identified. Some mutants showed additional phenotypes, such as increased sensitivity to UV irradiation, methyl methanesulfonate, and hydroxyurea, that were suggestive of defects in DNA repair and/or checkpoint mechanisms. We have cloned and characterized the gene of one complementation group, PIP3, whose product is necessary both for delaying entry into S phase or mitosis when cells are UV irradiated in G1 or G2 phase and for lowering the rate of ongoing DNA synthesis in the presence of methyl methanesulfonate. PIP3 turned out to be the MEC3 gene, previously identified as a component of the G2 DNA damage checkpoint. The finding that Mec3 is also required for the G1- and S-phase DNA damage checkpoints, together with the analysis of genetic interactions between a mec3 null allele and several conditional DNA replication mutations at the permissive temperature, suggests that Mec3 could be part of a mechanism coupling DNA replication with repair of DNA damage, and DNA primase might be involved in this process.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Replicação do DNA , Genes Fúngicos , RNA Nucleotidiltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular/genética , DNA Primase , Relação Dose-Resposta à Radiação , Teste de Complementação Genética , Genótipo , Hidroxiureia/farmacologia , Metanossulfonato de Metila/farmacologia , Mitose , Mutagênese , Mutagênicos/farmacologia , RNA Nucleotidiltransferases/genética , Fase S , Saccharomyces cerevisiae/crescimento & desenvolvimento , Temperatura , Raios UltravioletaRESUMO
The B subunit of the DNA polymerase (pol) alpha-primase complex executes an essential role at the initial stage of DNA replication in Saccharomyces cerevisiae and is phosphorylated in a cell cycle-dependent manner. In this report, we show that the four subunits of the yeast DNA polymerase alpha-primase complex are assembled throughout the cell cycle, and physical association between newly synthesized pol alpha (p180) and unphosphorylated B subunit (p86) occurs very rapidly. Therefore, B subunit phosphorylation does not appear to modulate p180.p86 interaction. Conversely, by depletion experiments and by using a yeast mutant strain, which produces a low and constitutive level of the p180 polypeptide, we found that formation of the p180.p86 subcomplex is required for B subunit phosphorylation.
Assuntos
DNA Polimerase II/metabolismo , Replicação do DNA , RNA Nucleotidiltransferases/metabolismo , Ciclo Celular , DNA Primase , Substâncias Macromoleculares , Peso Molecular , Fosforilação , Ligação Proteica , Saccharomyces cerevisiae/enzimologiaRESUMO
The in vivo function of the 34 kDa subunit of yeast replication protein A (RPA), encoded by the RFA2 gene, has been studied by analyzing the effect of Rpa34 depletion and by producing and characterizing rfa2 temperature-sensitive mutants. We show that unbalanced stoichiometry of the RPA subunits does not affect cell growth and cell cycle progression until the level of Rpa34 becomes rate-limiting, at which point cells arrest with a late S/G2 DNA content. Rpa34 is involved in DNA replication in vivo, since rfa2 ts mutants are defective in S phase progression and ARS plasmid stability, and rfa2 pol1 double mutants are non-viable. Moreover, when shifted to the restrictive temperature, about 50% of the rfa2 mutant cells rapidly die while traversing the S phase and the surviving cells arrest in late S/G2 at the RAD9 checkpoint. Finally, rfa2 mutant cells have a mutator and hyper-recombination phenotype and are more sensitive to hydroxyurea and methyl-methane-sulfonate than wild-type cells.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Glicosiltransferases/genética , Mutação , Fase S/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fatores de Transcrição , Proteínas de Bactérias/genética , Sequência de Bases , Ciclo Celular/genética , Morte Celular/genética , Divisão Celular/genética , DNA Polimerase II , Reparo do DNA , Replicação do DNA , Fase G2/genética , Dados de Sequência Molecular , Plasmídeos/química , Proteína de Replicação A , Proteínas Repressoras/genética , TemperaturaRESUMO
The yeast DNA polymerase alpha-primase B subunit functions in initiation of DNA replication. This protein is present in two forms, of 86 and 91 kDa, and the p91 polypeptide results from cell cycle-regulated phosphorylation of p86. The B subunit present in G1 arises by dephosphorylation of p91 while cells are exiting from mitosis, becomes phosphorylated in early S phase, and is competent and sufficient to initiate DNA replication. The B subunit transiently synthesized as a consequence of periodic transcription of the POL12 gene is phosphorylated no earlier than G2. Phosphorylation of the B subunit does not require execution of the CDC7-dependent step and ongoing DNA synthesis. We suggest that posttranslational modifications of the B subunit might modulate the role of DNA polymerase alpha-primase in DNA replication.
Assuntos
Ciclo Celular/fisiologia , RNA Nucleotidiltransferases/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Fosfatase Ácida , Western Blotting , DNA Primase , Replicação do DNA , Fase G1 , Expressão Gênica , Genótipo , Cinética , Substâncias Macromoleculares , Mutagênese , Fosforilação , Plasmídeos , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Solanum tuberosum/enzimologia , Fatores de TempoRESUMO
Replication factor A (RF-A) is a heterotrimeric single-stranded-DNA-binding protein which is conserved in all eukaryotes. Since the availability of conditional mutants is an essential step to define functions and interactions of RF-A in vivo, we have produced and characterized mutations in the RFA1 gene, encoding the p70 subunit of the complex in Saccharomyces cerevisiae. This analysis provides the first in vivo evidence that RF-A function is critical not only for DNA replication but also for efficient DNA repair and recombination. Moreover, genetic evidence indicate that p70 interacts both with the DNA polymerase alpha-primase complex and with DNA polymerase delta.
Assuntos
Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA/genética , Recombinação Genética , Saccharomyces cerevisiae/genética , Análise Mutacional de DNA , DNA Polimerase II/metabolismo , DNA Polimerase III , DNA Primase , DNA Fúngico/genética , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas Fúngicas/genética , RNA Nucleotidiltransferases/metabolismo , Proteína de Replicação ARESUMO
The four-subunit DNA polymerase alpha-primase complex is unique in its ability to synthesize DNA chains de novo, and some in vitro data suggest its involvement in initiation and elongation of chromosomal DNA replication, although direct in vivo evidence for a role in the initiation reaction is still lacking. The function of the B subunit of the complex is unknown, but the Saccharomyces cerevisiae POL12 gene, which encodes this protein, is essential for cell viability. We have produced different pol12 alleles by in vitro mutagenesis of the cloned gene. The in vivo analysis of our 18 pol12 alleles indicates that the conserved carboxy-terminal two-thirds of the protein contains regions that are essential for cell viability, while the more divergent NH2-terminal portion is partially dispensable. The characterization of the temperature-sensitive pol12-T9 mutant allele demonstrates that the B subunit is required for in vivo DNA synthesis and correct progression through S phase. Moreover, reciprocal shift experiments indicate that the POL12 gene product plays an essential role at the early stage of chromosomal DNA replication, before the hydroxyurea-sensitive step. A model for the role of the B subunit in initiation of DNA replication at an origin is presented.
Assuntos
Replicação do DNA , Genes Fúngicos , RNA Nucleotidiltransferases/metabolismo , Saccharomyces cerevisiae/enzimologia , Alelos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Western Blotting , Cromossomos Fúngicos/efeitos dos fármacos , DNA Primase , Homeostase , Humanos , Hidroxiureia/farmacologia , Cinética , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos BALB C/imunologia , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Insercional , Mutagênese Sítio-Dirigida , RNA Nucleotidiltransferases/análise , RNA Nucleotidiltransferases/biossíntese , Saccharomyces cerevisiae/genética , Deleção de Sequência , Homologia de Sequência de AminoácidosRESUMO
A new DNA polymerase activity was identified and purified to near homogeneity from extracts of mitotic and meiotic cells of the yeast Saccharomyces cerevisiae. This activity increased at least 5-fold during meiosis, and it was shown to be associated with a 68-kDa polypeptide as determined by SDS-polyacrylamide gel electrophoresis. This new DNA polymerase did not have any detectable 3'-->5' exonuclease activity and preferred small gapped DNA as a template-primer. The activity was inhibited by dideoxyribonucleoside 5'-triphosphates and N-ethylmaleimide but not by concentrations of aphidicolin which completely inhibit either DNA polymerases I (alpha), II (epsilon), or III (delta). Since no polypeptide(s) in the extensively purified DNA polymerase fractions cross-reacted with antibodies raised against yeast DNA polymerases I, II, and III, we called this enzyme DNA polymerase IV. The DNA polymerase IV activity increased at least 10-fold in a yeast strain overexpressing the gene product predicted from the YCR14C open-reading frame (identified on S. cerevisiae chromosome III and provisionally called POLX), while no activity was detected in a strain where POLX was deleted. These results strongly suggest that DNA polymerase IV is encoded by the POLX gene and is a probable homolog of mammalian DNA polymerase beta.
Assuntos
DNA Polimerase Dirigida por DNA/isolamento & purificação , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Animais , Sequência de Bases , DNA Polimerase I/genética , DNA Polimerase beta , Primers do DNA , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Humanos , Cinética , Magnésio , Dados de Sequência Molecular , Inibidores da Síntese de Ácido Nucleico , Cloreto de Potássio , Homologia de Sequência do Ácido Nucleico , Especificidade por SubstratoRESUMO
The POL1 gene, encoding DNA polymerase alpha (pol alpha) in Saccharomyces cerevisiae, is transiently transcribed during the cell cycle at the G1/S phase boundary. Here we show that yeast pol alpha is present at every stage of the cell cycle, and its level only slightly increases following the peak of POL1 transcription. POL1 mRNA synthesis driven by a GAL1 promoter can be completely abolished without affecting the growth rate of logarithmically growing yeast cultures for several cell divisions, although the amount of the pol alpha polypeptide drops below the physiological level. Moreover, alpha-factor-arrested cells can enter S phase and divide synchronously even if POL1 transcription is abolished. These results indicate that the level of yeast pol alpha is not rate limiting and de novo synthesis of the enzyme is not required for entrance into S phase.