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1.
Biol Pharm Bull ; 47(7): 1338-1344, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39048355

RESUMO

The initiation of DNA replication is tightly controlled by the licensing system that loads replicative DNA helicases onto replication origins to form pre-replicative complexes (pre-RCs) once per cell cycle. Cdc10-dependent transcript 1 (Cdt1) plays an essential role in the licensing reaction by recruiting mini-chromosome maintenance (MCM) complexes, which are eukaryotic replicative DNA helicases, to their origins via direct protein-protein interactions. Cdt1 interacts with other pre-RC components, the origin recognition complex, and the cell division cycle 6 (Cdc6) protein; however, the molecular mechanism by which Cdt1 functions in the MCM complex loading process has not been fully elucidated. Here, we analyzed the protein-protein interactions of recombinant Cdt1 and observed that Cdt1 self-associates via the central region of the molecule, which is inhibited by the endogenous licensing inhibitor, geminin. Mutation of two ß-strands of the winged-helix domain in the central region of Cdt1 attenuated its self-association but could still interact with other pre-RC components and DNA similarly to wild-type Cdt1. Moreover, the Cdt1 mutant showed decreased licensing activity in Xenopus egg extracts. Together, these results suggest that the self-association of Cdt1 is crucial for licensing.


Assuntos
Proteínas de Ciclo Celular , Geminina , Animais , Geminina/metabolismo , Geminina/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Replicação do DNA , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis , Domínios Proteicos , Xenopus , Humanos , Proteínas de Ligação a DNA
2.
Cancer Genomics Proteomics ; 21(2): 166-177, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38423594

RESUMO

BACKGROUND/AIM: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with dismal prognosis. Genomic instability due to defects in cell-cycle regulation/mitosis or deficient DNA-damage repair is a major driver of PDAC progression with clinical relevance. Deregulation of licensing of DNA replication leads to DNA damage and genomic instability, predisposing cells to malignant transformation. While overexpression of DNA replication-licensing factors has been reported in several human cancer types, their role in PDAC remains largely unknown. We aimed here to examine the expression and prognostic significance of the DNA replication-licensing factors chromatin licensing and DNA replication factor 1 (CDT1), cell-division cycle 6 (CDC6), minichromosome maintenance complex component 7 (MCM7) and also of the ubiquitin ligase regulator of CDT1, cullin 4A (CUL4A), in PDAC. MATERIALS AND METHODS: Expression levels of CUL4, CDT1, CDC6 and MCM7 were evaluated by immunohistochemistry in 76 formalin-fixed paraffin-embedded specimens of PDAC patients in relation to DNA-damage response marker H2AX, clinicopathological parameters and survival. We also conducted bioinformatics analysis of data from online available databases to corroborate our findings. RESULTS: CUL4A and DNA replication-licensing factors were overexpressed in patients with PDAC and expression of CDT1 positively correlated with H2AX. Expression of CUL4A and CDT1 positively correlated with lymph node metastasis. Importantly, elevated CUL4A expression was associated with reduced overall survival and was an independent indicator of poor prognosis on multivariate analysis. CONCLUSION: Our findings implicate CUL4A, CDT1, CDC6 and MCM7 in PDAC progression and identify CUL4A as an independent prognostic factor for this disease.


Assuntos
Adenocarcinoma , Neoplasias Pancreáticas , Humanos , Adenocarcinoma/genética , Ligases/genética , Ubiquitina , Neoplasias Pancreáticas/genética , Proteínas de Ciclo Celular/genética , DNA , Instabilidade Genômica , Proteínas Culina/genética , Proteínas Culina/metabolismo
3.
Biochem Biophys Res Commun ; 651: 30-38, 2023 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-36791496

RESUMO

Speckle-type pox virus and zinc finger (POZ) protein (SPOP), a substrate recognition receptor for the cullin-3/RING ubiquitin E3 complex, leads to the ubiquitination of >40 of its target substrates. Since a variety of point mutations in the substrate-binding domain of SPOP have been identified in cancers, including prostate and endometrial cancers, the pathological roles of those cancer-associated SPOP mutants have been extensively elucidated. In this study, we evaluated the cellular functions of wild-type SPOP in non-cancerous human keratinocyte-derived HaCaT cells expressing wild-type SPOP gene. SPOP knockdown using siRNA in HaCaT cells dramatically reduced cell growth and arrested their cell cycles at G1/S phase. The expression of DNA replication licensing factors CDT1 and CDC6 in HaCaT cells drastically decreased on SPOP knockdown as their translation was inhibited. CDT1 and CDC6 downregulation induced p21 expression without p53 activation. Our results suggest that SPOP is essential for DNA replication licensing in non-cancerous keratinocyte HaCaT cells.


Assuntos
Neoplasias do Endométrio , Células HaCaT , Masculino , Feminino , Humanos , Células HaCaT/metabolismo , Células HaCaT/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Replicação do DNA/genética , Ubiquitinação , Neoplasias do Endométrio/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo
4.
Biol Reprod ; 106(4): 730-740, 2022 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-34977916

RESUMO

Origin recognition complex subunit 4 (ORC4) is a DNA-binding protein required for DNA replication. During oocyte maturation, after the last oocyte DNA replication step and before zygotic DNA replication, the oocyte undergoes two meiotic cell divisions in which half the DNA is ejected in much smaller polar bodies. We previously demonstrated that ORC4 forms a cytoplasmic cage around the DNA that is ejected in both polar body extrusion (PBE) events. Here, we used ZP3 activated Cre to delete exon 7 of Orc4 during oogenesis to test how it affected both predicted functions of ORC4: its recently discovered role in PBE and its well-known role in DNA synthesis. Orc4 deletion severely reduced PBE. Almost half of Orc4-depleted germinal vesicle (GV) oocytes cultured in vitro were arrested before anaphase I (48%), and only 25% produced normal first polar bodies. This supports the role of ORC4 in PBE and suggests that transcription of the full-length Orc4 during oogenesis is required for efficient PBE. Orc4 deletion also abolished zygotic DNA synthesis. Fewer Orc4-depleted oocytes developed to the metaphase II (MII) stage, and after activation these oocytes were arrested at the two-cell stage without undergoing DNA synthesis. This confirms that transcription of full-length Orc4 after the primary follicle stage is required for zygotic DNA replication. The data also suggest that MII oocytes do not have a replication licensing checkpoint as cytokinesis progressed without DNA synthesis. Together, the data confirm that oocyte ORC4 is important for both PBE and zygotic DNA synthesis.


Assuntos
Complexo de Reconhecimento de Origem , Corpos Polares , Replicação do DNA , Meiose/genética , Oogênese/genética , Complexo de Reconhecimento de Origem/genética , Complexo de Reconhecimento de Origem/metabolismo
5.
Genes Cells ; 25(7): 475-482, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32294311

RESUMO

Stomata are tiny pores on plant leaves and stems surrounded by a pair of differentiated epidermal cells known as guard cells. Plants undergo guard cell differentiation in response to environmental cues, including atmospheric CO2 . To quantitatively evaluate stomatal development in response to elevated CO2 , imaging analysis of stomata was conducted using young cotyledons of Arabidopsis thaliana grown under ambient (380 ppm) and elevated (1,000 ppm) CO2 conditions. Our analysis revealed that treatment with 1,000 ppm CO2 did not affect stomatal numbers on abaxial sides of cotyledons but increased cotyledon area, resulting in decreased stomatal density, 7 days after germination. Interestingly, this treatment also perturbed the uniform distribution of stomata via excess satellite stomata and stomatal precursor cells. We used overexpression lines of the DNA replication licensing factor gene CDC6, a reported positive regulator of satellite stomata production. CDC6 overexpression decreased the speed of cotyledon expansion, even under treatment with 1,000 ppm CO2 , possibly by suppressing pavement cell maturation. In contrast, treatment with 1,000 ppm CO2 induced stomatal distribution changes in the overexpressor. These results suggest that treatment with 1,000 ppm CO2 enhances both cotyledon expansion and satellite stomata production via independent pathways, at least in young cotyledons of A. thaliana.


Assuntos
Arabidopsis/metabolismo , Dióxido de Carbono/metabolismo , Cotilédone/metabolismo , Estômatos de Plantas/metabolismo , Arabidopsis/embriologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cotilédone/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Estômatos de Plantas/citologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Regulação para Cima
6.
Genetics ; 211(3): 797-829, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30846544

RESUMO

During animal development, a single fertilized egg forms a complete organism with tens to trillions of cells that encompass a large variety of cell types. Cell cycle regulation is therefore at the center of development and needs to be carried out in close coordination with cell differentiation, migration, and death, as well as tissue formation, morphogenesis, and homeostasis. The timing and frequency of cell divisions are controlled by complex combinations of external and cell-intrinsic signals that vary throughout development. Insight into how such controls determine in vivo cell division patterns has come from studies in various genetic model systems. The nematode Caenorhabditis elegans has only about 1000 somatic cells and approximately twice as many germ cells in the adult hermaphrodite. Despite the relatively small number of cells, C. elegans has diverse tissues, including intestine, nerves, striated and smooth muscle, and skin. C. elegans is unique as a model organism for studies of the cell cycle because the somatic cell lineage is invariant. Somatic cells divide at set times during development to produce daughter cells that adopt reproducible developmental fates. Studies in C. elegans have allowed the identification of conserved cell cycle regulators and provided insights into how cell cycle regulation varies between tissues. In this review, we focus on the regulation of the cell cycle in the context of C. elegans development, with reference to other systems, with the goal of better understanding how cell cycle regulation is linked to animal development in general.


Assuntos
Caenorhabditis elegans/genética , Ciclo Celular , Regulação da Expressão Gênica no Desenvolvimento , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Diferenciação Celular
7.
Dev Cell ; 45(1): 101-113.e4, 2018 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-29576425

RESUMO

The circadian clock and cell cycle as separate pathways have been well documented in plants. Elucidating whether these two oscillators are connected is critical for understanding plant growth. We found that a slow-running circadian clock decelerates the cell cycle and, conversely, a fast clock speeds it up. The clock component TOC1 safeguards the G1-to-S transition and controls the timing of the mitotic cycle at early stages of leaf development. TOC1 also regulates somatic ploidy at later stages of leaf development and in hypocotyl cells. The S-phase is shorter and delayed in TOC1 overexpressing plants, which correlates with the diurnal repression of the DNA replication licensing gene CDC6 through binding of TOC1 to the CDC6 promoter. The slow cell-cycle pace in TOC1-ox also results in delayed tumor progression in inflorescence stalks. Thus, TOC1 sets the time of the DNA pre-replicative machinery to control plant growth in resonance with the environment.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Replicação do DNA , Mitose/fisiologia , Tumores de Planta/genética , Fatores de Transcrição/metabolismo , Agrobacterium tumefaciens/patogenicidade , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Tumores de Planta/microbiologia , Fatores de Transcrição/genética
8.
Cell Cycle ; 17(4): 492-505, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29261034

RESUMO

The minichromosome maintenance (MCM) complex, consisting of six subunits, Mcm2-7, is loaded onto replication origins through loading factors (origin recognition complex [ORC], Cdc6, and Cdt1) and forms an MCM double hexamer that licenses the initiation of DNA replication. Previous studies with Xenopus egg extracts showed that loading factors, especially Cdc6, dissociate from chromatin on MCM loading, but the molecular mechanism and physiological significance remain largely unknown. Using a cell-free system for MCM loading onto plasmid DNA in Xenopus egg extracts, we found that MCM loaded onto DNA prevents DNA binding of the loading factors ORC, Cdc6, and Cdt1. We further report that a peptide of the C-terminal region of MCM3 (MCM3-C), previously implicated in the initial association with ORC/Cdc6 in budding yeast, prevents ORC/Cdc6/Cdt1 binding to DNA in the absence of MCM loading. ATP-γ-S suppresses inhibitory activities of both the MCM loaded onto DNA and the MCM3-C peptide. Other soluble factors in the extract, but neither MCM nor Cdt1, are required for the activity. Conservation of the amino acid sequences of MCM3-C and its activity in vertebrates implies a novel negative autoregulatory mechanism that interferes with MCM loading in the vicinity of licensed origins to ensure proper origin licensing.


Assuntos
Replicação do DNA , Componente 3 do Complexo de Manutenção de Minicromossomo/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , DNA/química , DNA/metabolismo , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Componente 3 do Complexo de Manutenção de Minicromossomo/química , Componente 3 do Complexo de Manutenção de Minicromossomo/genética , Complexo de Reconhecimento de Origem/química , Complexo de Reconhecimento de Origem/metabolismo , Óvulo/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Ligação Proteica , Domínios Proteicos , Alinhamento de Sequência , Proteínas de Xenopus/química , Proteínas de Xenopus/genética
9.
Biochim Biophys Acta Mol Cell Res ; 1864(6): 987-996, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28161520

RESUMO

Colorectal cancer is one of the most common cancers in industrialised societies. Epidemiological studies, animal experiments, and randomized clinical trials have shown that dietary factors can influence all stages of colorectal carcinogenesis, from initiation through promotion to progression. Calcium is one of the factors with a chemoprophylactic effect in colorectal cancer. The aim of this study was to understand the molecular mechanisms of the anti-tumorigenic effects of extracellular calcium ([Ca2+]o) in colon cancer cells. Gene expression microarray analysis of colon cancer cells treated for 1, 4, and 24h with 2mM [Ca2+]o identified significant changes in expression of 1571 probe sets (ANOVA, p<10-5). The main biological processes affected by [Ca2+]o were DNA replication, cell division, and regulation of transcription. All factors involved in DNA replication-licensing were significantly downregulated by [Ca2+]o. Furthermore, we show that the calcium-sensing receptor (CaSR), a G protein-coupled receptor is a mediator involved in this process. To test whether these results were physiologically relevant, we fed mice with a standard diet containing low (0.04%), intermediate (0.1%), or high (0.9%) levels of dietary calcium. The main molecules regulating replication licensing were inhibited also in vivo, in the colon of mice fed high calcium diet. We show that among the mechanisms behind the chemopreventive effect of [Ca2+]o is inhibition of replication licensing, a process often deregulated in neoplastic transformation. Our data suggest that dietary calcium is effective in preventing replicative stress, one of the main drivers of cancer and this process is mediated by the calcium-sensing receptor.


Assuntos
Cálcio/metabolismo , Neoplasias Colorretais/genética , Replicação do DNA , Perfilação da Expressão Gênica , Células CACO-2 , Neoplasias Colorretais/patologia , Células HT29 , Humanos , RNA Mensageiro/genética
10.
Cell Cycle ; 16(5): 415-427, 2017 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-28135906

RESUMO

Cyclin G-associated kinase (GAK) harbors a consensus phosphorylation motif (Y412) for c-Src; however, its physiological significance remains elusive. Here, we show that GAK is phosphorylated by c-Src not only at Y412 but also at Y1149. An anti-GAK-pY412 antibody recognized the shifted band of GAK during M phase. Immunofluorescence (IF) showed that GAK-pY412/pY1149 signals were present in the nucleus during interphase, translocated to chromosomes at prophase and prometaphase, moved to centrosomes at metaphase, and finally translocated to chromosomes at the end of telophase, when nuclear membrane formation was almost complete. These subcellular movements of GAK resemble those of DNA licensing factors. Indeed, mass spectrometry identified mini-chromosome maintenance (MCM) 3, an essential component of the DNA licensing system, as one of the association partners of GAK; immunoprecipitation-mediated Western blotting confirmed their association in vivo. These results suggest that the c-Src_GAK_MCM axis plays an important role in cell cycle progression through control of the DNA replication licensing system.


Assuntos
Centrossomo/metabolismo , Cromatina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Telófase , Quinases da Família src/metabolismo , Anticorpos/metabolismo , Núcleo Celular/metabolismo , Proliferação de Células , Células HeLa , Humanos , Interfase , Componente 2 do Complexo de Manutenção de Minicromossomo/metabolismo , Componente 3 do Complexo de Manutenção de Minicromossomo/metabolismo , Mitose , Fosforilação , Ligação Proteica , Transporte Proteico , Fase S , Frações Subcelulares/enzimologia
11.
Persoonia ; 30: 106-25, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24027350

RESUMO

The recently recognised protein-coding genes MCM7 and TSR1 have shown significant promise for phylogenetic resolution within the Ascomycota and Basidiomycota, but have remained unexamined within other fungal groups (except for Mucorales). We designed and tested primers to amplify these genes across early-diverging fungal clades, with emphasis on the Kickxellomycotina, zygomycetous fungi with characteristic flared septal walls forming pores with lenticular plugs. Phylogenetic tree resolution and congruence with MCM7 and TSR1 were compared against those inferred with nuclear small (SSU) and large subunit (LSU) rRNA genes. We also combined MCM7 and TSR1 data with the rDNA data to create 3- and 4-gene trees of the Kickxellomycotina that help to resolve evolutionary relationships among and within the core clades of this subphylum. Phylogenetic inference suggests that Barbatospora, Orphella, Ramicandelaber and Spiromyces may represent unique lineages. It is suggested that these markers may be more broadly useful for phylogenetic studies among other groups of early-diverging fungi.

12.
Persoonia ; 23: 35-40, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20198159

RESUMO

Developing powerful phylogenetic markers is a key concern in fungal phylogenetics. Here we report degenerate primers that amplify the single-copy genes Mcm7 (MS456) and Tsr1 (MS277) across a wide range of Pezizomycotina (Ascomycota). Phylogenetic analyses of 59 taxa belonging to the Eurotiomycetes, Lecanoromycetes, Leotiomycetes, Lichinomycetes and Sordariomycetes, indicate the utility of these loci for fungal phylogenetics at taxonomic levels ranging from genus to class. We also tested the new primers in silico using sequences of Saccharomycotina, Taphrinomycotina and Basidiomycota to predict their potential of amplifying widely across the Fungi. The analyses suggest that the new primers will need no, or only minor sequence modifications to amplify Saccharomycotina, Taphrinomycotina and Basidiomycota.

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