RESUMO
The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.
Assuntos
Autoantígenos/genética , Proteínas Cromossômicas não Histona/genética , Histonas/genética , Autoantígenos/metabolismo , Centrômero , Proteína Centromérica A , Proteínas Cromossômicas não Histona/metabolismo , Histonas/metabolismo , Humanos , Cinetocoros , Escleroderma Sistêmico/genética , Terminologia como AssuntoRESUMO
Asymmetric cell division requires the orientation of mitotic spindles along the cell-polarity axis. In Drosophila neuroblasts, this involves the interaction of the proteins Inscuteable (Insc) and Partner of inscuteable (Pins). We report here that a human Pins-related protein, called LGN, is instead essential for the assembly and organization of the mitotic spindle. LGN is cytoplasmic in interphase cells, but associates with the spindle poles during mitosis. Ectopic expression of LGN disrupts spindle-pole organization and chromosome segregation. Silencing of LGN expression by RNA interference also disrupts spindle-pole organization and prevents normal chromosome segregation. We found that LGN binds the nuclear mitotic apparatus protein NuMA, which tethers spindles at the poles, and that this interaction is required for the LGN phenotype. Anti-LGN antibodies and the LGN-binding domain of NuMA both trigger microtubule aster formation in mitotic Xenopus egg extracts, and the NuMA-binding domain of LGN blocks aster assembly in egg extracts treated with taxol. Thus, we have identified a mammalian Pins homologue as a key regulator of spindle organization during mitosis.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Drosophila , Proteínas de Insetos/metabolismo , Proteínas Nucleares/metabolismo , Fuso Acromático/metabolismo , Proteínas de Xenopus , Animais , Anticorpos , Antígenos Nucleares , Sítios de Ligação/fisiologia , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Expressão Gênica/fisiologia , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Rim/citologia , Mamíferos , Microtúbulos/metabolismo , Mitose/fisiologia , Proteínas Associadas à Matriz Nuclear , Proteínas Nucleares/química , Oócitos/metabolismo , Ligação Proteica/fisiologia , RNA/farmacologia , Coelhos , XenopusRESUMO
Pin1 is an essential and conserved mitotic peptidyl-prolyl isomerase (PPIase) that is distinct from members of two other families of conventional PPIases, cyclophilins and FKBPs (FK-506 binding proteins). In response to their phosphorylation during mitosis, Pin1 binds and regulates members of a highly conserved set of proteins that overlaps with antigens recognized by the mitosis-specific monoclonal antibody MPM-2. Pin1 is here shown to be a phosphorylation-dependent PPIase that specifically recognizes the phosphoserine-proline or phosphothreonine-proline bonds present in mitotic phosphoproteins. Both Pin1 and MPM-2 selected similar phosphorylated serine-proline-containing peptides, providing the basis for the specific interaction between Pin1 and MPM-2 antigens. Pin1 preferentially isomerized proline residues preceded by phosphorylated serine or threonine with up to 1300-fold selectivity compared with unphosphorylated peptides. Pin1 may thus regulate mitotic progression by catalyzing sequence-specific and phosphorylation-dependent proline isomerization.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Mitose , Oligopeptídeos/metabolismo , Peptidilprolil Isomerase/metabolismo , Fosfoproteínas/metabolismo , Prolina/metabolismo , Isomerases de Aminoácido/metabolismo , Anticorpos Monoclonais , Sítios de Ligação , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ligação a DNA/metabolismo , Epitopos , Células HeLa , Proteínas de Choque Térmico/metabolismo , Humanos , Isomerismo , Modelos Moleculares , Peptidilprolil Isomerase de Interação com NIMA , Oligopeptídeos/química , Biblioteca de Peptídeos , Peptidilprolil Isomerase/química , Fosfoproteínas/química , Fosfoproteínas/imunologia , Fosforilação , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Proteínas de Ligação a TacrolimoRESUMO
LIS1, a microtubule-associated protein, is required for neuronal migration, but the precise mechanism of LIS1 function is unknown. We identified a LIS1 interacting protein encoded by a mouse homolog of NUDE, a nuclear distribution gene in A. nidulans and a multicopy suppressor of the LIS1 homolog, NUDF. mNudE is located in the centrosome or microtubule organizing center (MTOC), and interacts with six different centrosomal proteins. Overexpression of mNudE dissociates gamma-tubulin from the centrosome and disrupts microtubule organization. Missense mutations that disrupt LIS1 function block LIS1-mNudE binding. Moreover, misexpression of the LIS1 binding domain of mNudE in Xenopus embryos disrupts the architecture and lamination of the CNS. Thus, LIS1-mNudE interactions may regulate neuronal migration through dynamic reorganization of the MTOC.
Assuntos
Sistema Nervoso Central/embriologia , Sistema Nervoso Central/metabolismo , Centrossomo/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterase , Animais , Células COS , Embrião não Mamífero/anormalidades , Embrião não Mamífero/patologia , Proteínas Fúngicas/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , RNA Mensageiro/biossíntese , Homologia de Sequência de Aminoácidos , Especificidade por Substrato/fisiologia , Tubulina (Proteína)/metabolismo , Técnicas do Sistema de Duplo-Híbrido , XenopusRESUMO
BACKGROUND: Cyclin-dependent kinases (CDKs) are thought to initiate and coordinate cell division processes by sequentially phosphorylating key targets; in most cases these substrates remain unidentified. RESULTS: Using a screen that scores for phosphorylation of proteins, which were translated from pools of cDNA plasmids in vitro, by either phosphoepitope antibody recognition or electrophoretic mobility shifts, we have identified 20 mitotically phosphorylated proteins from Xenopus embryos, 15 of which have sequence similarity to other proteins. Of these proteins, five have previously been shown to be phosphorylated during mitosis (epithelial-microtubule associated protein-115, Oct91, Elongation factor 1gamma, BRG1 and Ribosomal protein L18A), five are related to proteins postulated to have roles in mitosis (epithelial-microtubule associated protein-115, Schizosaccharomyces pombe Cdc5, innercentrosome protein, BRG1 and the RNA helicase WM6), and nine are related to transcription factors (BRG1, negative co-factor 2alpha, Oct91, S. pombe Cdc5, HoxD1, Sox3, Vent2, and two isoforms of Xbr1b). Of 16 substrates tested, 14 can be directly phosphorylated in vitro by the mitotic CDK, cyclin B-Cdc2, although three of these may be physiological substrates of other kinases activated during mitosis. CONCLUSIONS: Examination of this broad set of mitotic phosphoproteins has allowed us to draw three conclusions about how the activation of CDKs regulates cell-cycle events. First, Cdc2 itself appears to directly phosphorylate most of the mitotic phosphoproteins. Second, during mitosis most of the substrates are phosphorylated more than once and a number may be targets of multiple kinases, suggesting combinatorial regulation. Third, the large fraction of mitotic phosphoproteins that are presumptive transcription factors, two of which have been previously shown to dissociate from DNA during mitosis, suggests that an important function of mitotic phosphorylation is to strip the chromatin of proteins associated with gene expression.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Embrião não Mamífero/fisiologia , Proteínas dos Microtúbulos/biossíntese , Fosfoproteínas/biossíntese , Animais , Proteína Quinase CDC2/metabolismo , Clonagem Molecular , Embrião não Mamífero/citologia , Epitopos/análise , Fertilização , Interfase , Proteínas dos Microtúbulos/isolamento & purificação , Mitose , Fosfopeptídeos/análise , Fosfopeptídeos/química , Fosfoproteínas/química , Fosfoproteínas/isolamento & purificação , Fosforilação , Plasmídeos , Biossíntese de Proteínas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Schizosaccharomyces/citologia , Schizosaccharomyces/fisiologia , XenopusAssuntos
Apoptose , Proteínas de Ciclo Celular/biossíntese , Ciclo Celular/genética , Clonagem Molecular/métodos , Transfecção/métodos , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Cisteína Endopeptidases/metabolismo , DNA Complementar , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/metabolismo , Feminino , Homeostase , Humanos , Interfase , Células Jurkat , Mitose , Oócitos , RNA/administração & dosagem , RNA/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Xenopus laevisRESUMO
Transcript depletion using small interfering RNA (siRNA) technology represents a potentially valuable technique for the treatment of cancer. However, delivering therapeutic quantities of siRNA into solid tumors by chemical transfection is not feasible, whereas viral vectors efficiently transduce many human tumor cell lines. Yet producing sufficient quantities of viral vectors that elicit acute and selective cytotoxicity remains a major obstacle for preclinical and clinical trials. Using the invertebrate Spodoptera frugiperda (Sf9) cell line, we were able to produce high titer stocks of cytotoxic recombinant adeno-associated virus (rAAV) that express short hairpin RNA (shRNA) and that efficiently deplete Hec1 (highly expressed in cancer 1), or Kntc2 (kinetochore-associated protein 2), a kinetochore protein directly involved in kinetochore microtubule interactions, chromosome congression and spindle checkpoint signaling. Depletion of Hec1 protein results in persistent spindle checkpoint activation followed by cell death. Because Hec1 expression and activity are only present in mitotic cells, non-dividing cells were not affected by rAAV treatment. On the basis of the results of screening 56 human tumor cell lines with three different serotype vectors, we used a tumor xenograft model to test the effects in vivo. The effects of the shHec1 vector were evident in sectioned and stained tumors. The experiments with rAAV-shRNA vectors demonstrate the utility of producing vectors in invertebrate cells to obtain sufficient concentrations and quantities for solid tumor therapy. This addresses an important requirement for cancer gene therapy, to produce cytotoxic vectors in sufficient quantities and concentrations to enable quantitative transduction and selective killing of solid tumor cells.
Assuntos
Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/uso terapêutico , Neoplasias/terapia , Proteínas Nucleares/genética , RNA Interferente Pequeno/uso terapêutico , Animais , Linhagem Celular Tumoral , Proteínas do Citoesqueleto , Citometria de Fluxo , Deleção de Genes , Engenharia Genética , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Humanos , Cinetocoros/metabolismo , Camundongos , Camundongos Mutantes , Camundongos Nus , Microscopia de Fluorescência , Neoplasias/metabolismo , Neoplasias Experimentais , Proteínas Nucleares/metabolismo , Transdução Genética/métodos , Transplante HeterólogoRESUMO
Pin1 is an essential protein that can peptidyl-prolyl-isomerize small phosphopeptides. It has been suggested that Pin1 regulates entry into mitosis by catalyzing the cis/trans-isomerization of prolines on critical protein substrates in response to phosphorylation. We show that Pin1 catalytically generates a conformational change on the mitotic phosphatase Cdc25, as assayed by limited protease digestion, differential reactivity to a phosphoserine-proline-directed monoclonal antibody (MPM-2), and by changes in Cdc25 enzymatic activity. Pin1 catalytically modifies the conformation of Cdc25 at stoichiometries less than 0.0005, and mutants of Pin1 in the prolyl isomerase domain are not active. We suggest that, although difficult to detect, phosphorylation-dependent conformational changes mediated by prolyl isomerization may play an important regulatory role in the cell cycle.
Assuntos
Peptidilprolil Isomerase/farmacologia , Fosfatases cdc25/efeitos dos fármacos , Animais , Proteína Quinase CDC2/efeitos dos fármacos , Proteína Quinase CDC2/metabolismo , Proteína Quinase CDC2/farmacologia , Domínio Catalítico/genética , Relação Dose-Resposta a Droga , Humanos , Cinética , Mutação , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/genética , Fosforilação , Prolina/efeitos dos fármacos , Conformação Proteica , Xenopus , Fosfatases cdc25/química , Fosfatases cdc25/metabolismoRESUMO
Several different subassemblies of DNA polymerase III holoenzyme can be purified from Escherichia coli. Toward the goal of understanding the functional significance of these subassemblies, we have used the gamma complex clamp loader and the beta ring to assemble each different polymerase onto DNA. Through use of radioactive labeled proteins, the subunit structure of each resulting processive polymerase has been determined. Use of DNA polymerase III core, the gamma complex, and beta results in a core-beta complex on DNA; the gamma complex is not incorporated into the structure. The addition of tau to the assembly reaction to form either core1-tau 2 or core2-tau 2 results in a more efficient polymerase and more stabile association of core-tau beta on DNA, although the gamma complex still does not remain on DNA. The gamma complex clamp loader was retained on DNA with the other subunits only if it was first assembled into the polymerase (Pol) III* structure. The clamp loader within Pol III* appeared to be capable of loading two beta clamps onto DNA for both core polymerases within Pol III*, consistent with the hypothesis that one replicase can simultaneously replicate both strands of a duplex chromosome. These findings extend those of an earlier study showing that distinctive polymerases can be assembled depending on the presence or absence of tau (Maki, S., and Kornberg, A. (1988) J. Biol. Chem. 263, 6561-6569). The significance of these distinct polymerases in separate paths of DNA metabolism is discussed.
Assuntos
Cromossomos Bacterianos , DNA Polimerase III/metabolismo , Replicação do DNA , DNA Bacteriano/metabolismo , Isoenzimas/metabolismo , Escherichia coli/genéticaRESUMO
The gamma complex (gamma delta delta' chi psi) subassembly of DNA polymerase III holoenzyme transfers the beta subunit onto primed DNA in a reaction which requires ATP hydrolysis. Once on DNA, beta is a "sliding clamp" which tethers the polymerase to DNA for highly processive synthesis. We have examined beta and the gamma complex to identify which subunit(s) hydrolyzes ATP. We find the gamma complex is a DNA dependent ATPase. The beta subunit, which lacks ATPase activity, enhances the gamma complex ATPase when primed DNA is used as an effector. Hence, the gamma complex recognizes DNA and couples ATP hydrolysis to clamp beta onto primed DNA. Study of gamma complex subunits showed no single subunit contained significant ATPase activity. However, the heterodimers, gamma delta and gamma delta', were both DNA-dependent ATPases. Only the gamma delta ATPase was stimulated by beta and was functional in transferring the beta from solution to primed DNA. Similarity in ATPase activity of DNA polymerase III holoenzyme accessory proteins to accessory proteins of phage T4 DNA polymerase and mammalian DNA polymerase delta suggests the basic strategy of chromosome duplication has been conserved throughout evolution.
Assuntos
Adenosina Trifosfatases/metabolismo , DNA Polimerase III/metabolismo , Replicação do DNA , DNA Viral/metabolismo , Bacteriófago phi X 174/genética , Colífagos/genética , DNA Helicases/metabolismo , Cinética , Substâncias Macromoleculares , Oligodesoxirribonucleotídeos/metabolismo , Poli A/metabolismo , Poli dA-dT/metabolismo , Especificidade por Substrato , Moldes Genéticos , TermodinâmicaRESUMO
The replicase of E. coli, DNA polymerase III holoenzyme, is tightly fastened to DNA by its ring-shaped beta sliding clamp. However, despite being clamped to DNA, the polymerase must rapidly cycle on and off DNA to synthesize thousands of Okazaki fragments on the lagging strand. This study shows that DNA polymerase III holoenzyme cycles from one DNA to another by a novel mechanism of partial disassembly of its multisubunit structure and then reassembly. Upon completing a template, the polymerase disengages from its beta clamp, hops off DNA, and reassociates with another beta clamp at a new primed site. The original beta clamp is left on DNA and may be harnessed by other machineries to coordinate their action with chromosome replication.
Assuntos
DNA Polimerase III/metabolismo , Replicação do DNA , DNA Bacteriano/biossíntese , Escherichia coli/metabolismo , DNA , Dano ao DNA , Primers do DNA/metabolismo , Escherichia coli/enzimologia , Substâncias Macromoleculares , Modelos Genéticos , Ligação Proteica , Conformação ProteicaRESUMO
DNA polymerase III holoenzyme (holoenzyme), the multiprotein replicase of Escherichia coli, is essentially unlimited in processive DNA synthesis. Processive activity can be reconstituted from two components. One component, the beta preinitiation complex, is a beta dimer clamped onto primed DNA. The beta preinitiation complex is formed by the five-protein gamma complex, which hydrolyzes ATP to chaperone beta onto primed DNA. The other component is the alpha epsilon polymerase. The alpha epsilon polymerase itself is not processive, but is endowed with extremely high processive activity upon assembly with the beta preinitiation complex. Here we examine the mechanism by which the beta preinitiation complex confers processivity onto the alpha epsilon polymerase. We find the beta preinitiation complex to be mobile on DNA. Diffusion of beta on DNA is specific to duplex DNA, is bidirectional, does not require ATP, and appears to diffuse linearly along the duplex. Furthermore, beta directly binds the alpha epsilon polymerase through contact with alpha, the DNA polymerase subunit. Hence, the high processivity of the holoenzyme is rooted in a "sliding clamp" of beta on DNA that tethers the polymerase to the primed template. Implications for transcription and translation are discussed.
Assuntos
DNA Polimerase III/metabolismo , DNA Polimerase III/química , DNA Polimerase III/isolamento & purificação , Replicação do DNA , DNA Bacteriano/metabolismo , Escherichia coli/enzimologia , Substâncias Macromoleculares , Complexos Multiproteicos , Plasmídeos , Ligação Proteica , Moldes GenéticosRESUMO
Phosphorylation of mitotic proteins on the Ser/Thr-Pro motifs has been shown to play an important role in regulating mitotic progression. Pin1 is a novel essential peptidyl-prolyl isomerase (PPIase) that inhibits entry into mitosis and is also required for proper progression through mitosis, but its substrate(s) and function(s) remain to be determined. Here we report that in both human cells and Xenopus extracts, Pin1 interacts directly with a subset of mitotic phosphoproteins on phosphorylated Ser/Thr-Pro motifs in a phosphorylation-dependent and mitosis-specific manner. Many of these Pin1-binding proteins are also recognized by the monoclonal antibody MPM-2, and they include the important mitotic regulators Cdc25, Myt1, Wee1, Plk1, and Cdc27. The importance of this Pin1 interaction was tested by constructing two Pin1 active site point mutants that fail to bind a phosphorylated Ser/Thr-Pro motif in mitotic phosphoproteins. Wild-type, but not mutant, Pin1 inhibits both mitotic division in Xenopus embryos and entry into mitosis in Xenopus extracts. We have examined the interaction between Pin1 and Cdc25 in detail. Pin1 not only binds the mitotic form of Cdc25 on the phosphorylation sites important for its activity in vitro and in vivo, but it also inhibits its activity, offering one explanation for the ability of Pin1 to inhibit mitotic entry. In a separate paper, we have shown that Pin1 is a phosphorylation-dependent PPIase that can recognize specifically the phosphorylated Ser/Thr-Pro bonds present in mitotic phosphoproteins. Thus, Pin1 likely acts as a general regulator of mitotic proteins that have been phosphorylated by Cdc2 and other mitotic kinases.
Assuntos
Mitose , Peptidilprolil Isomerase/metabolismo , Fosfoproteínas/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Sítios de Ligação , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Fase G2 , Células HeLa/metabolismo , Humanos , Mutação , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/genética , Peptidilprolil Isomerase/imunologia , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas/genética , Fosforilação , Xenopus/embriologia , Fosfatases cdc25RESUMO
During mitosis, chromatin is condensed into mitotic chromosomes and transcription is inhibited, processes that might be opposed by the chromatin remodeling activity of the SWI/SNF complexes. Brg1 and hBrm, which are components of human SWI/SNF (hSWI/SNF) complexes, were recently shown to be phosphorylated during mitosis. This suggested that phosphorylation might be used as a switch to modulate SWI/SNF activity. Using an epitope-tag strategy, we have purified hSWI/SNF complexes at different stages of the cell cycle, and found that hSWI/SNF was inactive in cells blocked in G2-M. Mitotic hSWI/SNF contained Brg1 but not hBrm, and was phosphorylated on at least two subunits, hSWI3 and Brg1. In vitro, active hSWI/SNF from asynchronous cells can be phosphorylated and inactivated by ERK1, and reactivated by dephosphorylation. hSWI/SNF isolated as cells traversed mitosis regained activity when its subunits were dephosphorylated either in vitro or in vivo. We propose that this transitional inactivation and reactivation of hSWI/SNF is required for formation of a repressed chromatin structure during mitosis and reformation of an active chromatin structure as cells leave mitosis.
Assuntos
Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Mitose/fisiologia , Fatores de Transcrição/metabolismo , Sequência de Bases , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Ciclo Celular/fisiologia , Linhagem Celular , DNA Helicases , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Substâncias Macromoleculares , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Plasmídeos/genética , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
The replicative polymerase of Escherichia coli, DNA polymerase III, consists of a three-subunit core polymerase plus seven accessory subunits. Of these seven, tau and gamma are products of one replication gene, dnaX. The shorter gamma is created from within the tau reading frame by a programmed ribosomal -1 frameshift over codons 428 and 429 followed by a stop codon in the new frame. Two temperature-sensitive mutations are available in dnaX. The 2016(Ts) mutation altered both tau and gamma by changing codon 118 from glycine to aspartate; the 36(Ts) mutation affected the activity only of tau because it altered codon 601 (from glutamate to lysine). Evidence which indicates that, of these two proteins, only the longer tau is essential includes the following. (i) The 36(Ts) mutation is a temperature-sensitive lethal allele, and overproduction of wild-type gamma cannot restore its growth. (ii) An allele which produced tau only could be substituted for the wild-type chromosomal gene, but a gamma-only allele could not substitute for the wild-type dnaX in the haploid state. Thus, the shorter subunit gamma is not essential, suggesting that tau can be substitute for the usual function(s) of gamma. Consistent with these results, we found that a functional polymerase was assembled from nine pure subunits in the absence of the gamma subunit. However, the possibility that, in cells growing without gamma, proteolysis of tau to form a gamma-like product in amounts below the Western blot (immunoblot) sensitivity level cannot be excluded.
Assuntos
DNA Polimerase III/genética , Escherichia coli/enzimologia , Genes Bacterianos , Alelos , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Clonagem Molecular , DNA Polimerase III/isolamento & purificação , DNA Polimerase III/metabolismo , DNA Bacteriano , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação , Mapeamento por Restrição , TemperaturaRESUMO
Human SIX1 (HSIX1) is a member of the Six class of homeodomain proteins implicated in muscle, eye, head, and brain development. To further understand the role of HSIX1 in the cell cycle and cancer, we developed an HSIX1-specific antibody to study protein expression at various stages of the cell cycle. Our previous work demonstrated that HSIX1 mRNA expression increases as cells exit S phase and that overexpression of HSIX1 can attenuate a DNA damage-induced G(2) cell cycle checkpoint. Overexpression of HSIX1 mRNA was observed in 44% of primary breast cancers and 90% of metastatic lesions. Now we demonstrate that HSIX1 is a nuclear phosphoprotein that becomes hyperphosphorylated at mitosis in both MCF7 cells and in Xenopus extracts. The pattern of phosphorylation observed in mitosis is similar to that seen by treating recombinant HSIX1 with casein kinase II (CK2) in vitro. Apigenin, a selective CK2 inhibitor, diminishes interphase and mitotic phosphorylation of HSIX1. Treatment of MCF7 cells with apigenin leads to a dose-dependent arrest at the G(2)/M boundary, implicating CK2, like HSIX1, in the G(2)/M transition. HSIX1 hyperphosphorylated in vitro by CK2 loses its ability to bind the MEF3 sites of the aldolase A promoter (pM), and decreased binding to pM is observed during mitosis. Because CK2 and HSIX1 have both been implicated in cancer and in cell cycle control, we propose that HSIX1, whose activity is regulated by CK2, is a relevant target of CK2 in G(2)/M checkpoint control and that both molecules participate in the same pathway whose dysregulation leads to cancer.
Assuntos
Ciclo Celular , Proteínas de Homeodomínio/metabolismo , Sequência de Aminoácidos , Animais , Caseína Quinase II , Humanos , Dados de Sequência Molecular , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Transdução de Sinais , XenopusRESUMO
An interesting property of the Escherichia coli DNA polymerase II is the stimulation in DNA synthesis mediated by the DNA polymerase III accessory proteins beta,gamma complex. In this paper we have studied the basis for the stimulation in pol II activity and have concluded that these accessory proteins stimulate pol II activity by increasing the processivity of the enzyme between 150- and 600-fold. As is the case with pol III, processive synthesis by pol II requires both beta,gamma complex and SSB protein. Whereas the intrinsic velocity of synthesis by pol II is 20-30 nucleotides per s with or without the accessory proteins, the processivity of pol II is increased from approximately five nucleotides to greater than 1600 nucleotides incorporated per template binding event. The effect of the accessory proteins on the rate of replication is far greater on pol III than on pol II; pol III holoenzyme is able to complete replication of circular single-stranded M13 DNA in less than 20 s, whereas pol II in the presence of the gamma complex and beta requires approximately 5 min. We have investigated the effect of beta,gamma complex proteins on bypass of a site-specific abasic lesion by E. coli DNA polymerases I, II, and III. All three polymerases are extremely inefficient at bypass of the abasic lesion. We find limited bypass by pol I with no change upon addition of accessory proteins. pol II also shows limited bypass of the abasic site, dependent on the presence of beta,gamma complex and SSB. pol III shows no significant bypass of the abasic site with or without beta,gamma complex.
Assuntos
DNA Polimerase III/metabolismo , DNA Polimerase II/metabolismo , Replicação do DNA , Autorradiografia , Bacteriófago phi X 174/metabolismo , DNA Viral/metabolismo , Eletroforese em Gel de Ágar , Escherichia coli/enzimologiaRESUMO
BACKGROUND: The high speed and processivity of replicative DNA polymerases reside in a processivity factor which has been shown to be a ring-shaped protein. This protein ("sliding clamp') encircles DNA and tethers the catalytic unit to the template. Although in eukaryotic, prokaryotic and bacteriophage-T4 systems, the processivity factors are ring-shaped, they assume different oligomeric states. The Escherichia coli clamp (the beta subunit) is active as a dimer while the eukaryotic and T4 phage clamps (PCNA and gp45, respectively) are active as trimers. The clamp can not assemble itself on DNA. Instead, a protein complex known as a clamp loader utilizes ATP to assemble the ring around the primer-template. This study compares properties of the human PCNA clamp with those of E. coli and T4 phage. RESULTS: The PCNA ring is a stable trimer down to a concentration below 100 nM (Kd approximately 21 nM). On DNA, the PCNA clamp slides freely and dissociates from DNA slowly (t1/2 approximately 24 min). beta is more stable in solution (Kd < 60 PM) and on DNA (t1/2 approximately 1 h) than PCNA which may be explained by its simpler oligomeric state. The T4 gp45 clamp is a much less stable trimer than PCNA (Kd approximately 250 nM) and requires association with the polymerase to stabilize it on DNA as observed previously. The consequence of this cooperation between clamp and polymerase is that upon finishing a template and dissociation of the polymerase from DNA, the gp45 clamp spontaneously dissociates from DNA without assistance. However, the greater stability of the PCNA and beta clamps on DNA necessitates an active process for their removal. The clamp loaders (RFC and gamma complex) were also capable of unloading their respective clamps from DNA in the presence of ATP. CONCLUSIONS: The stability of the different clamps in solution correlates with their stability on DNA. Thus, the low stability of the T4 clamp explains the inability to isolate gp45 on DNA. The stability of the PCNA and beta clamps predicts they will require an unloading factor to recycle them on and off DNA during replication. The clamp loaders of PCNA and beta double as clamp unloaders presumably for the purpose of clamp recycling.