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1.
Mol Cell ; 37(6): 753-67, 2010 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-20347419

RESUMO

The regulation of mitotic entry in somatic cells differs from embryonic cells, yet it is only for embryonic cells that we have a quantitative understanding of this process. To gain a similar insight into somatic cells, we developed a human cell extract system that recapitulates CDK1 activation and nuclear envelope breakdown in response to mitotic cyclins. As cyclin B concentrations increase, CDK1 activates in a three-stage nonlinear response, creating an ordering of substrate phosphorylations. This response is established by dual regulatory feedback loops involving WEE1/MYT1, which impose a cyclin B threshold, and CDC25, which allows CDK1 to escape the WEE1/MYT1 inhibition. This system also exhibits a complex response to cyclin A. Cyclin A promotes WEE1 phosphorylation to weaken the negative feedback loop and primes mitotic entry through cyclin B. This observation explains the requirement of both cyclin A and cyclin B to initiate mitosis in somatic cells.


Assuntos
Proteína Quinase CDC2/análise , Extratos Celulares/química , Mitose , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina A2/genética , Ciclina A2/metabolismo , Ciclina B/metabolismo , Ativação Enzimática , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Fosfotirosina/metabolismo , Ligação Proteica , Proteínas Tirosina Quinases/metabolismo , Especificidade por Substrato
2.
Proc Natl Acad Sci U S A ; 111(44): 15768-73, 2014 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-25324523

RESUMO

Rapid progression through the cell cycle and a very short G1 phase are defining characteristics of embryonic stem cells. This distinct cell cycle is driven by a positive feedback loop involving Rb inactivation and reduced oscillations of cyclins and cyclin-dependent kinase (Cdk) activity. In this setting, we inquired how ES cells avoid the potentially deleterious consequences of premature mitotic entry. We found that the pluripotency transcription factor Oct4 (octamer-binding transcription factor 4) plays an unappreciated role in the ES cell cycle by forming a complex with cyclin-Cdk1 and inhibiting Cdk1 activation. Ectopic expression of Oct4 or a mutant lacking transcriptional activity recapitulated delayed mitotic entry in HeLa cells. Reduction of Oct4 levels in ES cells accelerated G2 progression, which led to increased chromosomal missegregation and apoptosis. Our data demonstrate an unexpected nontranscriptional function of Oct4 in the regulation of mitotic entry.


Assuntos
Quinases Ciclina-Dependentes/metabolismo , Células-Tronco Embrionárias/metabolismo , Fase G2/fisiologia , Fator 3 de Transcrição de Octâmero/metabolismo , Animais , Proteína Quinase CDC2 , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Ciclinas/metabolismo , Células-Tronco Embrionárias/citologia , Ativação Enzimática/fisiologia , Fase G1/fisiologia , Células HeLa , Humanos , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo
3.
Nucleic Acids Res ; 37(3): 661-71, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19240147

RESUMO

The nucleotide sequence of DNA is the repository of hereditary information. Yet, it is now clear that the DNA itself plays an active role in regulating the ability of the cell to extract its information. Basic biological processes, including control of gene transcription, faithful DNA replication and segregation, maintenance of the genome and cellular differentiation are subject to the conformational and topological properties of DNA in addition to the regulation imparted by the sequence itself. How do these DNA features manifest such striking effects and how does the cell regulate them? In this review, we describe how misregulation of DNA topology can lead to cellular dysfunction. We then address how cells prevent these topological problems. We close with a discussion on recent theoretical advances indicating that the topological problems, themselves, can provide the cues necessary for their resolution by type-2 topoisomerases.


Assuntos
DNA Topoisomerases Tipo II/metabolismo , DNA/química , DNA/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico
4.
SLAS Discov ; 26(4): 547-559, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33780296

RESUMO

Recent advances in targeted protein degradation have enabled chemical hijacking of the ubiquitin-proteasome system to treat disease. The catalytic rate of cereblon (CRBN)-dependent bifunctional degradation activating compounds (BiDAC), which recruit CRBN to a chosen target protein, resulting in its ubiquitination and proteasomal degradation, is an important parameter to consider during the drug discovery process. In this work, an in vitro system was developed to measure the kinetics of BRD4 bromodomain 1 (BD1) ubiquitination by fitting an essential activator kinetic model to these data. The affinities between BiDACs, BD1, and CRBN in the binary complex, ternary complex, and full ubiquitination complex were characterized. Together, this work provides a new tool for understanding and optimizing the catalytic and thermodynamic properties of BiDACs.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Bioensaio , Proteínas de Ciclo Celular/metabolismo , Oxindóis/farmacologia , Ftalimidas/farmacologia , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Sistema Livre de Células/química , Sistema Livre de Células/metabolismo , Células HeLa , Humanos , Cinética , Oxindóis/síntese química , Ftalimidas/síntese química , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Ligação Proteica , Domínios Proteicos , Proteólise/efeitos dos fármacos , Termodinâmica , Fatores de Transcrição/química , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/efeitos dos fármacos
5.
BMC Mol Biol ; 8: 44, 2007 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-17531098

RESUMO

BACKGROUND: The genetic code imposes a dilemma for cells. The DNA must be long enough to encode for the complexity of an organism, yet thin and flexible enough to fit within the cell. The combination of these properties greatly favors DNA collisions, which can knot and drive recombination of the DNA. Despite the well-accepted propensity of cellular DNA to collide and react with itself, it has not been established what the physiological consequences are. RESULTS: Here we analyze the effects of recombined and knotted plasmids in E. coli using the Hin site-specific recombination system. We show that Hin-mediated DNA knotting and recombination (i) promote replicon loss by blocking DNA replication; (ii) block gene transcription; and (iii) cause genetic rearrangements at a rate three to four orders of magnitude higher than the rate for an unknotted, unrecombined plasmid. CONCLUSION: These results show that DNA reactivity leading to recombined and knotted DNA is potentially toxic and may help drive genetic evolution.


Assuntos
DNA Nucleotidiltransferases/metabolismo , DNA/química , DNA/metabolismo , Mutação , Conformação de Ácido Nucleico , Recombinação Genética , Replicon , DNA/genética , DNA Nucleotidiltransferases/genética , Replicação do DNA , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Reporter , Plasmídeos/genética , Plasmídeos/metabolismo , Replicon/genética , beta-Lactamases/genética , beta-Lactamases/metabolismo
6.
Mol Microbiol ; 58(1): 80-101, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16164551

RESUMO

The physiological role of topoisomerase III is unclear for any organism. We show here that the removal of topoisomerase III in temperature sensitive topoisomerase IV mutants in Escherichia coli results in inviability at the permissive temperature. The removal of topoisomerase III has no effect on the accumulation of catenated intermediates of DNA replication, even when topoisomerase IV activity is removed. Either recQ or recA null mutations, but not helD null or lexA3, partially rescued the synthetic lethality of the double topoisomerase III/IV mutant, indicating a role for topoisomerase III in recombination. We find a bias against deleting the gene encoding topoisomerase III in ruvC53 or DeltaruvABC backgrounds compared with the isogenic wild-type strains. The topoisomerase III RuvC double mutants that can be constructed are five- to 10-fold more sensitive to UV irradiation and mitomycin C treatment and are twofold less efficient in transduction efficiency than ruvC53 mutants. The overexpression of ruvABC allows the construction of the topoisomerase III/IV double mutant. These data are consistent with a role for topoisomerase III in disentangling recombination intermediates as an alternative to RuvABC to maintain the stability of the genome.


Assuntos
DNA Topoisomerases Tipo I/fisiologia , Proteínas de Escherichia coli/fisiologia , Escherichia coli/enzimologia , Recombinação Genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , DNA Helicases/genética , DNA Helicases/fisiologia , DNA Topoisomerases Tipo I/genética , DNA Bacteriano/metabolismo , DNA Catenado/análise , DNA Super-Helicoidal/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/fisiologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Deleção de Genes , Mutação , Recombinases Rec A/genética , Recombinases Rec A/fisiologia , RecQ Helicases , Serina Endopeptidases/genética , Temperatura , Transdução Genética
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