RESUMO
Here we discuss the important contributions that cell-free extracts have made to the study of complex biological processes. We provide a brief history of how cell-free extracts of frog eggs were developed to avoid many of the problems that can arise from the dilution and mixing of cellular components that typically occur when cell-free extracts are prepared. We briefly describe how Xenopus egg extracts have been fundamental to the study of many important cellular processes including DNA replication, cell cycle progression, nuclear protein import, nuclear assembly and chromosome organisation. We describe how, in particular, Xenopus egg extracts have made a major contributions to the study of DNA replication, by permitting the direct manipulation of proteins in a system that is extraordinarily faithful to the way that DNA replication occurs in the living embryo. Finally we consider how results obtained using Xenopus egg extracts are being translated to produce diagnostic reagents for cancer screening and diagnosis.
Assuntos
Núcleo Celular/metabolismo , Sistema Livre de Células , Replicação do DNA/fisiologia , Oócitos/metabolismo , Animais , XenopusRESUMO
Nuclear export of mRNAs is a crucial step in the regulation of gene expression, linking transcription in the nucleus to translation in the cytoplasm. Although important components of the mRNA export machinery are well characterized, such as transcription-export complexes TREX and TREX-2, recent work has shown that, in some instances, mammalian mRNA export can be selective and can regulate crucial biological processes such as DNA repair, gene expression, maintenance of pluripotency, haematopoiesis, proliferation and cell survival. Such findings show that mRNA export is an unexpected, yet potentially important, mechanism for the control of gene expression and of the mammalian transcriptome.
Assuntos
Transporte Ativo do Núcleo Celular , Regulação da Expressão Gênica , Mamíferos/genética , RNA Mensageiro/metabolismo , Animais , Citoplasma/metabolismo , Exodesoxirribonucleases/metabolismo , Humanos , Mamíferos/metabolismo , Neoplasias/metabolismo , Ribonucleoproteínas/metabolismoRESUMO
The nuclear phase of the gene expression pathway culminates in the export of mature messenger RNAs (mRNAs) to the cytoplasm through nuclear pore complexes. GANP (germinal- centre associated nuclear protein) promotes the transfer of mRNAs bound to the transport factor NXF1 to nuclear pore complexes. Here, we demonstrate that GANP, subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex, promotes selective nuclear export of a specific subset of mRNAs whose transport depends on NXF1. Genome-wide gene expression profiling showed that half of the transcripts whose nuclear export was impaired following NXF1 depletion also showed reduced export when GANP was depleted. GANP-dependent transcripts were highly expressed, yet short-lived, and were highly enriched in those encoding central components of the gene expression machinery such as RNA synthesis and processing factors. After injection into Xenopus oocyte nuclei, representative GANP-dependent transcripts showed faster nuclear export kinetics than representative transcripts that were not influenced by GANP depletion. We propose that GANP promotes the nuclear export of specific classes of mRNAs that may facilitate rapid changes in gene expression.
Assuntos
Acetiltransferases/fisiologia , Núcleo Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , RNA Mensageiro/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular Tumoral , Humanos , Proteínas de Transporte Nucleocitoplasmático/fisiologia , RNA Mensageiro/classificação , Proteínas de Ligação a RNA/fisiologia , XenopusRESUMO
In this article, we discuss the significance of DNA replication proteins in human disease. There is a broad range of mutations in genes encoding replication proteins, which result in several distinct clinical disorders that share common themes. One group of replication proteins, the MCMs, has emerged as effective biomarkers for early detection of a range of common cancers. They offer practical and theoretical advantages over other replication proteins and have been developed for widespread clinical use.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Replicação do DNA , Doenças Genéticas Inatas/genética , DNA Polimerase gama , DNA Polimerase Dirigida por DNA/genética , Humanos , Mutação , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/patologiaRESUMO
Messenger RNA (mRNA) export from the nucleus is essential for eukaryotic gene expression. Here we identify a transcript-selective nuclear export mechanism affecting certain human transcripts, enriched for functions in genome duplication and repair, controlled by inositol polyphosphate multikinase (IPMK), an enzyme catalyzing inositol polyphosphate and phosphoinositide turnover. We studied transcripts encoding RAD51, a protein essential for DNA repair by homologous recombination (HR), to characterize the mechanism underlying IPMK-regulated mRNA export. IPMK depletion or catalytic inactivation selectively decreases RAD51 protein abundance and the nuclear export of RAD51 mRNA, thereby impairing HR. Recognition of a sequence motif in the untranslated region of RAD51 transcripts by the mRNA export factor ALY requires IPMK. Phosphatidylinositol (3,4,5)-trisphosphate (PIP3), an IPMK product, restores ALY recognition in IPMK-depleted cell extracts, suggesting a mechanism underlying transcript selection. Our findings implicate IPMK in a transcript-selective mRNA export pathway controlled by phosphoinositide turnover that preserves genome integrity in humans.
Assuntos
Transporte Ativo do Núcleo Celular/genética , Instabilidade Genômica , Fosfotransferases (Aceptor do Grupo Álcool)/genética , RNA Mensageiro/genética , Linhagem Celular Tumoral , Núcleo Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genoma Humano , Recombinação Homóloga/genética , Humanos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositóis/metabolismo , Fosforilação/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , RNA Mensageiro/metabolismo , Transdução de SinaisRESUMO
Export of messenger RNA (mRNA) from the nucleus to the cytoplasm is a critical step in the gene expression pathway of eukaryotic cells. Here, we report the functional and structural characterization of the mammalian TREX-2 complex and show how it links transcription/processing with nuclear mRNA export. Mammalian TREX-2 is based on a germinal-centre associated nuclear protein (GANP) scaffold to which ENY2, PCID2 and centrins bind and depletion of any of these components inhibits mRNA export. The crystal structure of the GANP:ENY2 complex shows that two ENY2 chains interact directly with GANP, but they have different orientations from those observed on yeast Sac3. GANP is required to recruit ENY2 to nuclear pore complexes (NPCs), but ENY2 is not necessary to recruit GANP, which requires both its CID and MCM3AP domains, together with nucleoporin Nup153. GANP and ENY2 associate with RNA polymerase II and inhibition of mRNA processing redistributes GANP from NPCs into nuclear foci indicating that mammalian TREX-2 is associated with transcription. Thus, we implicate TREX-2 as an integral component of the mammalian mRNA export machinery where it links transcription and nuclear export by facilitating the transfer of mature mRNPs from the nuclear interior to NPCs.
Assuntos
Acetiltransferases/química , Núcleo Celular/genética , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , RNA Mensageiro/metabolismo , Transcrição Gênica , Acetiltransferases/análise , Acetiltransferases/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Cristalografia por Raios X , Exodesoxirribonucleases/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Modelos Moleculares , Dados de Sequência Molecular , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/análise , Fosfoproteínas/metabolismo , Transporte de RNA , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
The first differentiation event in mammalian development gives rise to the blastocyst, consisting of two cell lineages that have also segregated in how the cell cycle is structured. Pluripotent cells of the inner cell mass divide mitotically to retain a diploid DNA content, but the outer trophoblast cells can amplify their genomes more than 500-fold by undergoing multiple rounds of DNA replication, completely bypassing mitosis. Central to this striking divergence in cell cycle control is the E3 ubiquitin-ligase activity of the anaphase-promoting complex or cyclosome (APC/C). Extended suppression of APC/C activity during interphase of mouse pluripotent cells promotes rapid cell cycle progression by allowing stabilization of cyclins, whereas unopposed APC/C activity during S phase of mouse trophoblast cells triggers proteasomal-mediated degradation of geminin and giant cell formation. While differential APC/C activity might govern the atypical cell cycles observed in pre-implantation mouse embryos, geminin is a critical APC/C substrate that: (1) escapes degradation in pluripotent cells to maintain expression of Oct4, Sox2 and Nanog; and (2) mediates specification and endoreduplication when targeted for ectopic destruction in trophoblast. Thus, in contrast to trophoblast giant cells that lack geminin, geminin is preserved in both mouse pluripotent cells and non-endoreduplicating human cytotrophoblast cells.
Assuntos
Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Ciclina A2/metabolismo , Ciclina B1/metabolismo , Células-Tronco Embrionárias/metabolismo , Endorreduplicação , Geminina , Humanos , Interfase , Camundongos , Mitose , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteínas/antagonistas & inibidores , Proteínas/genética , Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Trofoblastos/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Geminin is an essential cell-cycle protein that is only present from S phase to early mitosis in metazoan somatic cells. Genetic ablation of geminin in the mouse results in preimplantation embryonic lethality because pluripotent cells fail to form and all cells differentiate to trophoblast. Here we show that geminin is present in G1 phase of mouse pluripotent cells in contrast to somatic cells, where anaphase-promoting complex/cyclosome (APC/C)-mediated proteasomal destruction removes geminin in G1. Silencing geminin directly or by depleting the APC/C inhibitor Emi1 causes loss of stem cell identity and trophoblast differentiation of mouse embryonal carcinoma and embryonic stem cells. Depletion of cyclins A2 or B1 does not induce this effect, even though both of these APC/C substrates are also present during G1 of pluripotent cells. Crucially, geminin antagonizes the chromatin-remodeling protein Brg1 to maintain expression of Oct4, Sox2, and Nanog. Our results define a pluripotency pathway by which suppressed APC/C activity protects geminin from degradation in G1, allowing sustained expression of core pluripotency factors. Collectively, these findings link the cell cycle to the pluripotent state but also raise an unexplained paradox: How is cell-cycle progression possible in pluripotent cells when oscillations of key regulatory proteins are lost?
Assuntos
Proteínas de Ciclo Celular/metabolismo , Fase G1 , Proteínas de Homeodomínio/metabolismo , Proteínas Nucleares/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Animais , Diferenciação Celular , Ciclina A2/metabolismo , Ciclina B1/metabolismo , DNA Helicases/metabolismo , Células-Tronco de Carcinoma Embrionário/citologia , Células-Tronco Embrionárias/citologia , Geminina , Camundongos , Proteína Homeobox Nanog , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
MCM3 acetylase (MCM3AP) and germinal-centre associated nuclear protein (GANP) are transcribed from the same locus and are therefore confused in databases because the MCM3 acetylase DNA sequence is contained entirely within the much larger GANP sequence and the entire MCM3AP sequence is identical to the carboxy terminus of GANP. Thus, the MCM3AP and GANP genes are read in the same reading frame and MCM3AP is an N-terminally truncated region of GANP. However, we show here that MCM3AP and GANP are different proteins, occupying different locations in the cell and transcribed from different promoters. Intriguingly, a promoter for MCM3AP lies within an intron of GANP. This report is an interesting example in nature of two separate gene products from the same locus that perform two entirely different functions in the cell. Therefore, to avoid further confusion, they should now be referred to as separate but overlapping genes.
Assuntos
Acetiltransferases/genética , Homologia de Genes , Bases de Dados Genéticas , Loci Gênicos , Genoma Humano , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Íntrons , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Análise de Sequência de DNA , Transcrição GênicaRESUMO
Nuclear export of mRNPs is mediated by transport factors such as NXF1 that bind mRNPs and mediate their translocation through the central channel of nuclear pores (NPC) using transient interactions with FG-nucleoporins. A number of nuclear factors enhance the efficiency of this process by concentrating mRNPs at the nuclear face of the pores. Although this enhancement has been explored mainly with the yeast TREX-2 complex, recent work has indicated that mammalian cells employ GANP (Germinal-centre Associated Nuclear Protein) for efficient mRNP nuclear export and for efficient recruitment of NXF1-containing mRNPs to NPCs. GANP is constructed from several domains that show local homology to FG-nucleoporins, the yeast mRNA export factor Sac3p and the mammalian MCM3 acetyltransferase. Whereas yeast TREX-2 is located primarily at nuclear pores, some GANP is located in the nuclear interior in addition to that found at the pores. GANP depletion inhibits bulk mRNA export, resulting in retention of mRNPs and NXF1 in punctate foci within the nucleoplasm, consistent with GANP's being an integral component of the mammalian mRNA export machinery. Here, we discuss the model for GANP function presented in our recent paper and its implications for the mechanism of mRNA export in mammalian cells.
Assuntos
Acetiltransferases/metabolismo , Poro Nuclear/metabolismo , Transporte de RNA/fisiologia , RNA Mensageiro/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Mamíferos , Modelos Biológicos , Poro Nuclear/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismoRESUMO
Bulk nuclear export of messenger ribonucleoproteins (mRNPs) through nuclear pore complexes (NPCs) is mediated by NXF1. It binds mRNPs through adaptor proteins such as ALY and SR splicing factors and mediates translocation through the central NPC transport channel via transient interactions with FG nucleoporins. Here, we show that mammalian cells require GANP (germinal center-associated nuclear protein) for efficient mRNP nuclear export and for efficient recruitment of NXF1 to NPCs. Separate regions of GANP show local homology to FG nucleoporins, the yeast mRNA export factor Sac3p, and the mammalian MCM3 acetyltransferase. GANP interacts with both NXF1 and NPCs and partitions between NPCs and the nuclear interior. GANP depletion inhibits mRNA export, with retention of mRNPs and NXF1 in punctate foci within the nucleus. The GANP N-terminal region that contains FG motifs interacts with the NXF1 FG-binding domain. Overexpression of this GANP fragment leads to nuclear accumulation of both poly(A)(+)RNA and NXF1. Treatment with transcription inhibitors redistributes GANP from NPCs into foci throughout the nucleus. These results establish GANP as an integral component of the mammalian mRNA export machinery and suggest a model whereby GANP facilitates the transfer of NXF1-containing mRNPs to NPCs.
Assuntos
Acetiltransferases/metabolismo , Centro Germinativo/metabolismo , Proteínas Nucleares/metabolismo , RNA Mensageiro/metabolismo , Acetiltransferases/antagonistas & inibidores , Acetiltransferases/genética , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Mamíferos , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/antagonistas & inibidores , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Fosfoproteínas/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: Promising stool-based screening tests for colorectal carcinoma (CRC) rely on detection of exfoliated colonocytes or their contents. However, methods of colonocyte retrieval have not been studied systematically and current approaches are restricted by low yields. We examined colonocyte numbers in stool wash fractions and assessed the suitability of retrieved cells for immunocytochemistry for minichromosome maintenance protein 2 (MCM2), a marker of the proliferative deregulation that characterizes malignancy. METHODS: Colonocyte numbers were accurately quantified in 129 wash fractions derived from 18 stools, comparing the mucus retained by a 125-microm filter (F fraction) with the fine and coarse content in the filtrate (S and P fractions, respectively). MCM2 immunocytochemistry was done on sections of fibrin clot containing filter-derived mucus, obtained from stools of eight independent subjects. RESULTS: Total colonocyte yield in the F fraction (mean, 433.8 per 100 microL) was higher than in the S (140.3) and P (204.6) fractions (P = 0.004 and 0.03, respectively) due to increased numbers of morphologically abnormal cells, which predominantly represented malignant cells in samples from CRC patients. Several thousand abnormal cells could be obtained from stool-derived mucus in all CRC patients, an order of magnitude greater than numbers in subjects without CRC. Median MCM2 labeling index in abnormal cells was 50% (range, 30-60%) in CRC patients and 0% in subjects without CRC. Cells in clot sections were well preserved and not obscured by fecal debris. CONCLUSIONS: Isolation of stool-derived mucus is technically straightforward and can improve the performance of protein-based and/or nucleic acid-based approaches to CRC screening.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias Colorretais/diagnóstico , Fezes/citologia , Mucosa Intestinal/citologia , Proteínas de Ciclo Celular/análise , Detecção Precoce de Câncer , Fezes/química , Fibrinogênio/análise , Humanos , Imuno-Histoquímica/métodos , Componente 2 do Complexo de Manutenção de Minicromossomo , Muco/química , Proteínas Nucleares/análise , Estatísticas não ParamétricasRESUMO
PURPOSE: Early detection of anal intraepithelial neoplasia (AIN) and anal squamous cell carcinoma (SCC) by screening will improve clinical outcome. Assessment of anal cytology samples using routine Papanicolaou testing suffers from shortcomings in sensitivity and/or specificity, suggesting that screening tests based on biomarkers may be of value. We tested the suitability in this context of minichromosome maintenance (MCM) proteins, accurate markers of the deregulated cell cycle entry that characterizes malignancy and premalignancy. EXPERIMENTAL DESIGN: We undertook an initial immunohistochemical study of 54 anal tissue samples and validated our findings using an independent prospective cohort study of 235 anal cytology samples from 144 subjects. RESULTS: In the progression from normal anal epithelium through AIN to SCC, there was increasing expression of MCM2 and MCM5, including in the superficial epithelial third, the source of the majority of cells collected by anal swab. The median labeling indices (LI) for MCM2 and MCM5 in the superficial third of AIN2/3 and SCCs combined were 90.2% and 84.0%, respectively. MCM LIs in the superficial layers were significantly greater than LIs for Ki67, an alternative marker of cell cycle entry (P<0.0001). By immunocytochemistry using a mixture of anti-MCM2 and anti-MCM5 antibodies, immunopositive cells were readily identified in anal cytology samples, even at low magnification. MCM testing showed sensitivity for AIN2/3 of 84% (95% confidence interval, 75,93) and for AIN1/viral changes of 76% (68, 84), with overall specificity (for any lesion) of 77% (64, 90). CONCLUSIONS: MCMs are promising biomarkers for improving detection of AIN and SCC in anal cytology samples.
Assuntos
Neoplasias do Ânus/diagnóstico , Carcinoma de Células Escamosas/diagnóstico , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Adulto , Idoso , Neoplasias do Ânus/metabolismo , Carcinoma de Células Escamosas/metabolismo , Progressão da Doença , Feminino , Humanos , Técnicas Imunoenzimáticas , Antígeno Ki-67/metabolismo , Masculino , Pessoa de Meia-Idade , Componente 2 do Complexo de Manutenção de Minicromossomo , Estudos Prospectivos , Sensibilidade e EspecificidadeRESUMO
The extracellular matrix (ECM) can induce chemotherapy resistance via AKT-mediated inhibition of apoptosis. Here, we show that loss of the ECM protein TGFBI (transforming growth factor beta induced) is sufficient to induce specific resistance to paclitaxel and mitotic spindle abnormalities in ovarian cancer cells. Paclitaxel-resistant cells treated with recombinant TGFBI protein show integrin-dependent restoration of paclitaxel sensitivity via FAK- and Rho-dependent stabilization of microtubules. Immunohistochemical staining for TGFBI in paclitaxel-treated ovarian cancers from a prospective clinical trial showed that morphological changes of paclitaxel-induced cytotoxicity were restricted to areas of strong expression of TGFBI. These data show that ECM can mediate taxane sensitivity by modulating microtubule stability.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Neoplasias Ovarianas/metabolismo , Paclitaxel/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Adesão Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Centrossomo/efeitos dos fármacos , Centrossomo/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas da Matriz Extracelular/deficiência , Feminino , Fibronectinas/metabolismo , Inativação Gênica/efeitos dos fármacos , Humanos , Integrinas/metabolismo , Mitose/efeitos dos fármacos , Modelos Biológicos , Neoplasias Ovarianas/patologia , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Fator de Crescimento Transformador beta/deficiência , Tubulina (Proteína)/metabolismoRESUMO
In multicellular eukaryotes, geminin prevents overreplication of DNA in proliferating cells. Here, we show that genetic ablation of geminin in the mouse prevents formation of inner cell mass (ICM) and causes premature endoreduplication at eight cells, rather than 32 cells. All cells in geminin-deficient embryos commit to the trophoblast cell lineage and consist of trophoblast giant cells (TGCs) only. Geminin is also down-regulated in TGCs of wild-type blastocysts during S and gap-like phases by proteasome-mediated degradation, suggesting that loss of geminin is part of the mechanism regulating endoreduplication.
Assuntos
Blastocisto/citologia , Proteínas de Ciclo Celular/metabolismo , Mamíferos/embriologia , Proteínas Nucleares/metabolismo , Células-Tronco Pluripotentes/fisiologia , Animais , Biomarcadores/metabolismo , Blastocisto/metabolismo , Fator de Transcrição CDX2 , Proteínas de Ciclo Celular/genética , Inibidor de Quinase Dependente de Ciclina p57/genética , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Desenvolvimento Embrionário , Geminina , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Leupeptinas/farmacologia , Camundongos , Camundongos Mutantes , Proteínas Nucleares/genética , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Inibidores de Proteassoma , Fase S/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Trofoblastos/citologiaRESUMO
Metazoans limit origin firing to once per cell cycle by oscillations in cyclin-dependent kinases and the replication licensing inhibitor geminin. Geminin inhibits pre-replication complex assembly by preventing Cdt1 from recruiting the minichromosome maintenance proteins to chromatin. Geminin depletion results in genomic over-replication in Drosophila and human cell lines. Here, we show that loss of geminin affects other cell cycle-dependent events in addition to DNA replication. Geminin inactivation causes centrosome overduplication without passage through mitosis in human normal and cancer cells. Centrosomes are microtubule-organizing centres that are duplicated during S phase and have an important role in the fidelity of chromosome transmission by nucleating the mitotic spindle. Consistent with this, geminin-depleted cells show multiple mitotic defects, including multipolar spindles, when driven into mitosis by checkpoint abrogation. These results show that the consequences of geminin loss exceed its immediate role in DNA replication and extend to promoting chromosome mis-segregation in mitosis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrossomo/metabolismo , Segregação de Cromossomos/fisiologia , Mitose/fisiologia , Fuso Acromático/metabolismo , Cafeína/farmacologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Núcleo Celular/metabolismo , Ciclina A/metabolismo , Geminina , Células HCT116 , Humanos , Mitose/efeitos dos fármacos , Fenótipo , Ploidias , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
The homomultimeric archaeal mini-chromosome maintenance (MCM) complex serves as a simple model for the analogous heterohexameric eukaryotic complex. Here we investigate the organization and orientation of the MCM complex of the hyperthermophilic archaeon Sulfolobus solfataricus (Sso) on model DNA substrates. Sso MCM binds as a hexamer and slides on the end of a 3'-extended single-stranded DNA tail of a Y-shaped substrate; binding is oriented so that the motor domain of the protein faces duplex DNA. Two candidate beta-hairpin motifs within the MCM monomer have partially redundant roles in DNA binding. Notably, however, conserved basic residues within these motifs have nonequivalent roles in the helicase activity of MCM. On the basis of these findings, we propose a model for the mechanism of the helicase activity of MCM and note parallels with SV40 T antigen.
Assuntos
Proteínas Arqueais/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas Nucleares/metabolismo , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/química , Humanos , Proteína 1 de Manutenção de Minicromossomo/química , Proteína 1 de Manutenção de Minicromossomo/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/química , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Alinhamento de Sequência , Soluções/química , Sulfolobus solfataricus/químicaRESUMO
Multiple conserved mechanisms limit DNA replication to once per cell cycle. One vital level of control focuses on the loading of the heterohexameric ring of minichromosome maintenance proteins (MCMs) onto chromatin in the hierarchical assembly of the pre-replication complex at origins of replication. An essential role in proliferation for MCMs and their regulators makes them potentially important biomarkers for routine clinical use in cancer detection and prognosis.
Assuntos
Biomarcadores Tumorais , Replicação do DNA , Proteínas de Ligação a DNA/química , Neoplasias/genética , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Humanos , Microscopia de Fluorescência , Modelos Biológicos , Neoplasias/diagnóstico , Neoplasias/patologia , PrognósticoRESUMO
Geminin inhibits DNA replication by preventing Cdt1 from loading minichromosome maintenance (MCM) proteins onto DNA. The present study has investigated whether the frequency of geminin expression predicts clinical outcome in breast cancer. Immunohistochemistry was used first to examine geminin expression in normal and malignant breast tissue (n = 67). Correlations with cell-cycle parameters, pathological features, and clinical outcome were then determined using an invasive breast carcinoma tissue microarray (n = 165). Breast carcinomas were scanned for mutations (n = 61) and copy number imbalances (n = 241) of the geminin gene. Finally, the cell cycle distribution of geminin in breast cancer cells was investigated in vivo and in vitro. Despite a putative tumour suppressor function, it was found that increased geminin expression is a powerful independent indicator of adverse prognosis in invasive breast cancer. Both poor overall survival (p = 0.0002) and the development of distant metastases (p = 0.005) are predicted by high geminin expression, which performs better in this patient cohort than traditional factors currently used to determine prognosis and appropriate therapy. No mutations or deletions of the geminin gene and no evidence that a high frequency of protein expression is related to gene amplification were found. It is shown that geminin is expressed from S to M phase in breast carcinoma tissue and cell lines, disappearing at the metaphase--anaphase transition. While MCM proteins identify all non-quiescent cells, geminin identifies the sub-fraction that have entered S phase, but not exited mitosis, thereby indicating the rate of cell-cycle progression. It is suggested that this explains its unexpected value as a prognostic marker in breast cancer.