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1.
Elife ; 92020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32808929

RESUMO

Genome replication is initiated from specific origin sites established by dynamic events. The Origin Recognition Complex (ORC) is necessary for orchestrating the initiation process by binding to origin DNA, recruiting CDC6, and assembling the MCM replicative helicase on DNA. Here we report five cryoEM structures of the human ORC (HsORC) that illustrate the native flexibility of the complex. The absence of ORC1 revealed a compact, stable complex of ORC2-5. Introduction of ORC1 opens the complex into several dynamic conformations. Two structures revealed dynamic movements of the ORC1 AAA+ and ORC2 winged-helix domains that likely impact DNA incorporation into the ORC core. Additional twist and pinch motions were observed in an open ORC conformation revealing a hinge at the ORC5·ORC3 interface that may facilitate ORC binding to DNA. Finally, a structure of ORC was determined with endogenous DNA bound in the core revealing important differences between human and yeast origin recognition.


Assuntos
Complexo de Reconhecimento de Origem/química , Estrutura Secundária de Proteína , Microscopia Crioeletrônica
3.
Elife ; 62017 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-28112645

RESUMO

Binding of the Origin Recognition Complex (ORC) to origins of replication marks the first step in the initiation of replication of the genome in all eukaryotic cells. Here, we report the structure of the active form of human ORC determined by X-ray crystallography and cryo-electron microscopy. The complex is composed of an ORC1/4/5 motor module lobe in an organization reminiscent of the DNA polymerase clamp loader complexes. A second lobe contains the ORC2/3 subunits. The complex is organized as a double-layered shallow corkscrew, with the AAA+ and AAA+-like domains forming one layer, and the winged-helix domains (WHDs) forming a top layer. CDC6 fits easily between ORC1 and ORC2, completing the ring and the DNA-binding channel, forming an additional ATP hydrolysis site. Analysis of the ATPase activity of the complex provides a basis for understanding ORC activity as well as molecular defects observed in Meier-Gorlin Syndrome mutations.


Assuntos
Adenosina Trifosfatases/química , Complexo de Reconhecimento de Origem/química , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica
4.
EMBO J ; 33(6): 605-20, 2014 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-24566989

RESUMO

Eukaryotic DNA replication initiates from multiple replication origins. To ensure each origin fires just once per cell cycle, initiation is divided into two biochemically discrete steps: the Mcm2-7 helicase is first loaded into prereplicative complexes (pre-RCs) as an inactive double hexamer by the origin recognition complex (ORC), Cdt1 and Cdc6; the helicase is then activated by a set of "firing factors." Here, we show that plasmids containing pre-RCs assembled with purified proteins support complete and semi-conservative replication in extracts from budding yeast cells overexpressing firing factors. Replication requires cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK). DDK phosphorylation of Mcm2-7 does not by itself promote separation of the double hexamer, but is required for the recruitment of firing factors and replisome components in the extract. Plasmid replication does not require a functional replication origin; however, in the presence of competitor DNA and limiting ORC concentrations, replication becomes origin-dependent in this system. These experiments indicate that Mcm2-7 double hexamers can be precursors of replication and provide insight into the nature of eukaryotic DNA replication origins.


Assuntos
Replicação do DNA/fisiologia , Ativação Enzimática/fisiologia , Proteínas de Manutenção de Minicromossomo/metabolismo , Complexos Multiproteicos/fisiologia , Origem de Replicação/fisiologia , Proteínas de Ciclo Celular/metabolismo , Espectrometria de Massas , Modelos Biológicos , Modelos Moleculares , Fosforilação , Plasmídeos/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales
5.
Artigo em Inglês | MEDLINE | ID: mdl-23838438

RESUMO

DNA replication is tightly controlled in eukaryotic cells to ensure that an exact copy of the genetic material is inherited by both daughter cells. Oscillating waves of cyclin-dependent kinase (CDK) and anaphase-promoting complex/cyclosome (APC/C) activities provide a binary switch that permits the replication of each chromosome exactly once per cell cycle. Work from several organisms has revealed a conserved strategy whereby inactive replication complexes are assembled onto DNA during periods of low CDK and high APC activity but are competent to execute genome duplication only when these activities are reversed. Periods of high CDK and low APC/C serve an essential function by blocking reassembly of replication complexes, thereby preventing rereplication. Higher eukaryotes have evolved additional CDK-independent mechanisms for preventing rereplication.


Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Ciclo Celular/fisiologia , Quinases Ciclina-Dependentes/metabolismo , Replicação do DNA/fisiologia , Eucariotos/fisiologia , Modelos Biológicos , Origem de Replicação/fisiologia , Saccharomyces cerevisiae/fisiologia , Schizosaccharomyces/fisiologia
6.
J Biol Chem ; 287(25): 21561-9, 2012 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-22544748

RESUMO

Antimitotic spindle poisons are among the most important chemotherapeutic agents available. However, precocious mitotic exit by mitotic slippage limits the cytotoxicity of spindle poisons. The MAD2-binding protein p31(comet) is implicated in silencing the spindle assembly checkpoint after all kinetochores are attached to spindles. In this study, we report that the levels of p31(comet) and MAD2 in different cell lines are closely linked with susceptibility to mitotic slippage. Down-regulation of p31(comet) increased the sensitivity of multiple cancer cell lines to spindle poisons, including nocodazole, vincristine, and Taxol. In the absence of p31(comet), lower concentrations of spindle poisons were required to induce mitotic block. The delay in checkpoint silencing was induced by an accumulation of mitotic checkpoint complexes. The increase in the duration of mitotic block after p31(comet) depletion resulted in a dramatic increase in mitotic cell death upon challenge with spindle poisons. Significantly, cells that are normally prone to mitotic slippage and resistant to spindle disruption-mediated mitotic death were also sensitized after p31(comet) depletion. These results highlight the importance of p31(comet) in checkpoint silencing and its potential as a target for antimitotic therapies.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/metabolismo , Citostáticos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Cinetocoros/metabolismo , Proteínas Nucleares/metabolismo , Fuso Acromático/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Células HeLa , Células Hep G2 , Humanos , Proteínas Nucleares/genética , Fuso Acromático/genética
7.
Mol Cancer Ther ; 10(5): 784-94, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21430130

RESUMO

Genotoxic stress such as ionizing radiation halts entry into mitosis by activation of the G(2) DNA damage checkpoint. The CHK1 inhibitor 7-hydroxystaurosporine (UCN-01) can bypass the checkpoint and induce unscheduled mitosis in irradiated cells. Precisely, how cells behave following checkpoint abrogation remains to be defined. In this study, we tracked the fates of individual cells after checkpoint abrogation, focusing in particular on whether they undergo mitotic catastrophe. Surprisingly, while a subset of UCN-01-treated cells were immediately eliminated during the first mitosis after checkpoint abrogation, about half remained viable and progressed into G(1). Both the delay of mitotic entry and the level of mitotic catastrophe were dependent on the dose of radiation. Although the level of mitotic catastrophe was specific for different cell lines, it could be promoted by extending the mitosis. In supporting this idea, weakening of the spindle-assembly checkpoint, by either depleting MAD2 or overexpressing the MAD2-binding protein p31(comet), suppressed mitotic catastrophe. Conversely, delaying of mitotic exit by depleting either p31(comet) or CDC20 tipped the balance toward mitotic catastrophe. These results underscore the interplay between the level of DNA damage and the effectiveness of the spindle-assembly checkpoint in determining whether checkpoint-abrogated cells are eliminated during mitosis.


Assuntos
Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Fase G2/efeitos dos fármacos , Mitose/efeitos dos fármacos , Mitose/genética , Inibidores de Proteínas Quinases/farmacologia , Estaurosporina/análogos & derivados , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Citocinese/efeitos dos fármacos , Citocinese/genética , Citocinese/efeitos da radiação , Dano ao DNA/efeitos da radiação , Fase G2/genética , Fase G2/efeitos da radiação , Raios gama , Células HCT116 , Células HeLa , Humanos , Camundongos , Mitose/efeitos da radiação , Células NIH 3T3 , Fuso Acromático , Estaurosporina/farmacologia
8.
J Biol Chem ; 283(23): 15716-23, 2008 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-18400748

RESUMO

Although cells can exit mitotic block aberrantly by mitotic slippage, they are prevented from becoming tetraploids by a p53-dependent postmitotic checkpoint. Intriguingly, disruption of the spindle-assembly checkpoint also compromises the postmitotic checkpoint. The precise mechanism of the interplay between these two pivotal checkpoints is not known. We found that after prolonged nocodazole exposure, the postmitotic checkpoint was facilitated by p53. We demonstrated that although disruption of the mitotic block by a MAD2-binding protein promoted slippage, it did not influence the activation of p53. Both p53 and its downstream target p21(CIP1/WAF1) were activated at the same rate irrespective of whether the spindle-assembly checkpoint was enforced or not. The accelerated S phase entry, as reflected by the premature accumulation of cyclin E relative to the activation of p21(CIP1/WAF1), is the reason for the uncoupling of the postmitotic checkpoint. In support of this hypothesis, forced premature mitotic exit with a specific CDK1 inhibitor triggered DNA replication without affecting the kinetics of p53 activation. Finally, replication after checkpoint bypass was boosted by elevating the level of cyclin E. These observations indicate that disruption of the spindle-assembly checkpoint does not directly influence p53 activation, but the shortening of the mitotic arrest allows cyclin E-CDK2 to be activated before the accumulation of p21(CIP1/WAF1). These data underscore the critical relationship between the spindle-assembly checkpoint and the postmitotic checkpoint in safeguarding chromosomal stability.


Assuntos
Ciclina E/metabolismo , Fase S/fisiologia , Fuso Acromático/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Instabilidade Cromossômica/efeitos dos fármacos , Instabilidade Cromossômica/fisiologia , Cromossomos Humanos/metabolismo , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Replicação do DNA/fisiologia , Células HeLa , Humanos , Proteínas Mad2 , Nocodazol/farmacologia , Poliploidia , Proteínas Repressoras/metabolismo , Fase S/efeitos dos fármacos , Moduladores de Tubulina/farmacologia
9.
Cell Cycle ; 7(10): 1449-61, 2008 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-18418077

RESUMO

Spindle-disrupting agents and CDK inhibitors are important cancer therapeutic agents. Spindle toxins activate the spindle-assembly checkpoint and lead to sustained activation of CDK1. Different published results indicate that CDK1 activity is either important or dispensable for the cytotoxicity associated with spindle disruption. Using live cell imaging and various approaches that uncoupled mitotic events, we show that apoptosis was induced by both prolonged nocodazole treatment as well as by inhibition of CDK1 activity after a transient nocodazole block. However, distinct mechanisms are involved in the two types of cell death. The massive apoptosis triggered by nocodazole treatment requires the continuous activation of cyclin B1-CDK1 and is antagonized by premature mitotic slippage. By contrast, apoptosis induced by nocodazole followed by CDK inhibitors occurred after rereplication and multipolar mitosis of the subsequent cell cycle. The presence of dual mechanisms of cytotoxicity mediated by spindle disruption and CDK inhibition may reconcile the various apparent inconsistent published results. These data underscore the essential role of cyclin B1-CDK1 as the basis of apoptosis during mitotic arrest, and the role of mitotic slippage and abnormal mitosis for apoptosis at later stages.


Assuntos
Apoptose/fisiologia , Proteína Quinase CDC2/antagonistas & inibidores , Mitose/fisiologia , Nocodazol/farmacologia , Fuso Acromático/efeitos dos fármacos , Moduladores de Tubulina/farmacologia , Apoptose/efeitos dos fármacos , Ativação Enzimática/fisiologia , Citometria de Fluxo , Células HeLa , Humanos , Immunoblotting , Mutagênese Sítio-Dirigida , Oligonucleotídeos
10.
Nucleic Acids Res ; 35(4): e22, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17234679

RESUMO

RNA interference (RNAi) by means of short hairpin RNA (shRNA) has developed into a powerful tool for loss-of-function analysis in mammalian cells. The principal problem in RNAi experiments is off-target effects, and the most vigorous demonstration of the specificity of shRNA is the rescue of the RNAi effects with a shRNA-resistant target gene. This presents its own problems, including the unpredictable relative expression of shRNA and rescue cDNA in individual cells, and the difficulty in generating stable cell lines. In this report, we evaluated the plausibility of combining the expression of shRNA and rescue cDNA in the same vector. In addition to facilitate the validation of shRNA specificity, this system also considerably simplifies the generation of shRNA-expressing cell lines. Since the compensatory cDNA is under the control of an inducible promoter, stable shRNA-expressing cells can be generated before the knockdown phenotypes are studied by conditionally turning off the rescue protein. Conversely, the rescue protein can be activated after the endogenous protein is completely repressed. This approach is particularly suitable when prolonged expression of either the shRNA or the compensatory cDNA is detrimental to cell growth. This system allows a convenient one-step validation of shRNA and generation of stable shRNA-expressing cells.


Assuntos
Interferência de RNA , RNA não Traduzido/biossíntese , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Ciclina A/antagonistas & inibidores , Ciclina A/genética , DNA Complementar/biossíntese , DNA Complementar/genética , Engenharia Genética/métodos , Vetores Genéticos , Células HeLa , Humanos , Proteínas Mad2 , Fenótipo , Plasmídeos/genética , RNA não Traduzido/química , RNA não Traduzido/genética , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética
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