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Eukaryotic cells coordinate chromosome duplication by the assembly of protein complexes at origins of DNA replication by sequential binding of member proteins of the origin recognition complex (ORC), CDC6, and minichromosome maintenance (MCM) proteins. These pre-replicative complexes (pre-RCs) are activated by cyclin-dependent kinases and DBF4/CDC7 kinase. Here, we carried out a comprehensive yeast two-hybrid screen to establish sequential interactions between two individual proteins of the mouse pre-RC that are probably required for the initiation of DNA replication. The studies revealed multiple interactions among ORC subunits and MCM proteins as well as interactions between individual ORC and MCM proteins. In particular CDC6 was found to bind strongly to ORC1 and ORC2, and to MCM7 proteins. DBF4 interacts with the subunits of ORC as well as with MCM proteins. It was also demonstrated that CDC7 binds to different ORC and MCM proteins. CDC45 interacts with ORC1 and ORC6, and weakly with MCM3, -6, and -7. The three subunits of the single-stranded DNA binding protein RPA show interactions with various ORC subunits as well as with several MCM proteins. The data obtained by yeast two-hybrid analysis were paradigmatically confirmed in synchronized murine FM3A cells by immunoprecipitation of the interacting partners. Some of the interactions were found to be cell-cycle-dependent; however, most of them were cell-cycle-independent. Altogether, 90 protein-protein interactions were detected in this study, 52 of them were found for the first time in any eukaryotic pre-RC. These data may help to understand the complex interplay of the components of the mouse pre-RC and should allow us to refine its structural architecture as well as its assembly in real time. 相似文献
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Control of the yeast cell cycle is associated with assembly/disassembly of the Cdc28 protein kinase complex 总被引:31,自引:0,他引:31
The Saccharomyces cerevisiae gene CDC28 encodes a protein kinase required for progression from G1 to S phase in the cell cycle. We present evidence that the active form of the Cdc28 protein kinase is a complex of approximately 160 kd containing an endogenous substrate, p40, and possibly other polypeptides. This complex phosphorylates p40 and exogenous histone H1 in vitro. Cell cycle arrest during G1 results in inactivation of the protein kinase accompanied by the disassembly of the complex. Furthermore, assembly of the complex is regulated during the cell cycle, reaching a maximum during G1. Partial complexes thought to be intermediates in the assembly process phosphorylate histone H1 but not p40. Addition of soluble factors to these partial complexes in vitro restores p40 phosphorylation and causes the complex to increase to the mature size. A model is presented in which p40 phosphorylation is required during G1 for cells to initiate a new cell cycle. 相似文献
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The Mu transpososome can distinguish between proximal and distal DNA during the selection of a site for transposition. This phenomenon, termed target immunity, involves MuA-stimulated removal of MuB oligomers from sites near the Mu genome. Using a combination of ensemble and single-molecule fluorescence methods, we show that the MuA tetramer can stably associate with the DNA-bound MuB oligomer and is more efficient than monomeric MuA at stimulating the dissociation of MuB from DNA. In addition, we demonstrate that DNA looping is essential for efficient disassembly of the MuB oligomer. We propose a model in which the MuA tetramer forms a multivalent complex with the MuB oligomer and catalyzes the processive removal of MuB from DNA. 相似文献
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An important aspect of cell behaviour is that DNA replication happens only once per cell cycle. Replicated DNA is unable to re-replicate until cell division has occurred. Unreplicated DNA is in a replication-competent or 'licensed' state. The ability to replicate is lost in S phase and regained following passage through mitosis. Recent evidence has implicated an MCM (minichromosome maintenance) protein complex and the Cdc6 protein in determining replication competence. Regeneration of replication competence upon passage through mitosis entails changes in protein kinase activity, of which the MCMs are a likely target. Features of the mechanism that restricts DNA replication to once per cell cycle appear to be conserved throughout eukaryotes. 相似文献
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Incomplete mitotic spindle disassembly causes lethality in budding yeast. To determine why spindle disassembly is required for cell viability, we used live-cell microscopy to analyze a double mutant strain containing a conditional mutant and a deletion mutant compromised for the kinesin-8 and anaphase-promoting complex-driven spindle-disassembly pathways (td-kip3 and doc1Δ, respectively). Under nonpermissive conditions, spindles in td-kip3 doc1Δ cells could break apart but could not disassemble completely. These cells could exit mitosis and undergo cell division. However, the daughter cells could not assemble functional, bipolar spindles in the ensuing mitosis. During the formation of these dysfunctional spindles, centrosome duplication and separation, as well as recruitment of key midzone-stabilizing proteins all appeared normal, but microtubule polymerization was nevertheless impaired and these spindles often collapsed. Introduction of free tubulin through episomal expression of α- and β-tubulin or introduction of a brief pulse of the microtubule-depolymerizing drug nocodazole allowed spindle assembly in these td-kip3 doc1Δ mutants. Therefore we propose that spindle disassembly is essential for regeneration of the intracellular pool of assembly-competent tubulin required for efficient spindle assembly during subsequent mitoses of daughter cells. 相似文献
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DNA strand scission and ADP-ribosyltransferase activity during murine erythroleukemia cell differentiation 总被引:1,自引:0,他引:1
The accumulation of DNA strand breaks and activation of ADP-ribosyltransferase (ADPRT) have recently been associated with cellular differentiation. Murine erythroleukemia (MEL) cells undergo erythropoietic differentiation when exposed to dimethyl sulfoxide (Me2SO) and several studies have suggested that DNA strand scission induced by this agent is a prerequisite for expression of the differentiated phenotype. Me2SO induction of MEL cells has also been associated with increases in ADPRT activity in one study, but not in another. We have monitored the effects of Me2SO on DNA strand breaks in preformed and replicating MEL cell DNA. The results clearly demonstrate that DNA fragmentation is not detectable during Me2SO induction of MEL differentiation, even in the presence of 3-aminobenzamide, an inhibitor of ADPRT. Further, these results are consistent with an absence of detectable changes in both endogenous and total potential ADPRT activity during Me2SO-induced MEL differentiation. These findings would argue against Me2SO induction of DNA strand scission and ADPRT in MEL cells undergoing differentiation. 相似文献
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The origin recognition complex (ORC) plays a central role in the initiation of DNA replication in eukaryotic cells. It interacts with origins of DNA replication in chromosomal DNA and recruits additional replication proteins to form functional initiation complexes. These processes have not been well characterized at the biochemical level except in the case of Saccharomyces cerevisiae ORC. We report here the expression, purification, and initial characterization of Schizosaccharomyces pombe ORC (SpORC) containing six recombinant subunits. Purified SpORC binds efficiently to the ars1 origin of DNA replication via the essential Nterminal domain of the SpOrc4 subunit which contains nine AT-hook motifs. Competition binding experiments demonstrated that SpORC binds preferentially to DNA molecules rich in AT-tracts, but does not otherwise exhibit a high degree of sequence specificity. The complex is capable of binding to multiple sites within the ars1 origin of DNA replication with similar affinities, indicating that the sequence requirements for origin recognition in S. pombe are significantly less stringent than in S. cerevisiae. We have also demonstrated that SpORC interacts directly with Cdc18p, an essential fission yeast initiation protein, and recruits it to the ars1 origin in vitro. Recruitment of Cdc18p to chromosomal origins is a likely early step in the initiation of DNA replication in vivo. These data indicate that the purified recombinant SpORC retains at least two of its primary biological functions and that it will be useful for the eventual reconstitution of the initiation reaction with purified proteins. 相似文献
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Nuclear pore complex assembly through the cell cycle: regulation and membrane organization 总被引:1,自引:0,他引:1
In eukaryotes, all macromolecules traffic between the nucleus and the cytoplasm through nuclear pore complexes (NPCs), which are among the largest supramolecular assemblies in cells. Although their composition in yeast and metazoa is well characterized, understanding how NPCs are assembled and form the pore through the double membrane of the nuclear envelope and how both processes are controlled still remains a challenge. Here, we summarize what is known about the biogenesis of NPCs throughout the cell cycle with special focus on the membrane reorganization and the regulation that go along with NPC assembly. 相似文献
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DNA damage to the germline genome must be accurately repaired to ensure transmission of intact genetic information to following generations. Meiosis presents challenges to the DNA damage response (DDR) because it universally requires changes to chromosome structure that can affect DNA repair outcomes. We report the existence of a meiotic DDR at chromosome axes that results in chromatin remodeling, synaptonemal complex disassembly, and axis separation in response to irradiation at late pachytene stages in C. elegans. The axis component HTP-3 is required for germline acquisition of H2AacK5, an axis-specific chromatin mark that is DNA damage responsive. Irradiated wild-types show reduction of H2AacK5 and axis separation that are dependent on the acetyltransferase MYS-1/TIP60. Restoration of H2AacK5 levels requires ATM-1 kinase and correlates with resynapsis. We propose that the meiotic DDR involves early chromatin remodeling at chromosome axes to dismantle structures promoting interhomolog recombination and facilitate efficient nonhomolog-based repair before pachytene exit. 相似文献
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The origin recognition complex in silencing, cell cycle progression, and DNA replication. 总被引:22,自引:1,他引:21
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This report describes the isolation of ORC5, the gene encoding the fifth largest subunit of the origin recognition complex, and the properties of mutants with a defective allele of ORC5. The orc5-1 mutation caused temperature-sensitive growth and, at the restrictive temperature, caused cell cycle arrest. At the permissive temperature, the orc5-1 mutation caused an elevated plasmid loss rate that could be suppressed by additional tandem origins of DNA replication. The sequence of ORC5 revealed a potential ATP binding site, making Orc5p a candidate for a subunit that mediates the ATP-dependent binding of ORC to origins. Genetic interactions among orc2-1 and orc5-1 and other cell cycle genes provided further evidence for a role for the origin recognition complex (ORC) in DNA replication. The silencing defect caused by orc5-1 strengthened previous connections between ORC and silencing, and combined with the phenotypes caused by orc2 mutations, suggested that the complex itself functions in both processes. 相似文献
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Komamura-Kohno Y Karasawa-Shimizu K Saitoh T Sato M Hanaoka F Tanaka S Ishimi Y 《The FEBS journal》2006,273(6):1224-1239
MCM4, a subunit of a putative replicative helicase, is phosphorylated during the cell cycle, at least in part by cyclin-dependent kinases (CDK), which play a central role in the regulation of DNA replication. However, detailed characterization of the phosphorylation of MCM4 remains to be performed. We examined the phosphorylation of human MCM4 at Ser3, Thr7, Thr19, Ser32, Ser54, Ser88 and Thr110 using anti-phosphoMCM4 sera. Western blot analysis of HeLa cells indicated that phosphorylation of MCM4 at these seven sites can be classified into two groups: (a) phosphorylation that is greatly enhanced in the G2 and M phases (Thr7, Thr19, Ser32, Ser54, Ser88 and Thr110), and (b) phosphorylation that is firmly detected during interphase (Ser3). We present data indicating that phosphorylation at Thr7, Thr19, Ser32, Ser88 and Thr110 in the M phase requires CDK1, using a temperature-sensitive mutant of mouse CDK1, and phosphorylation at sites 3 and 32 during interphase requires CDK2, using a dominant-negative mutant of human CDK2. Based on these results and those from in vitro phosphorylation of MCM4 with CDK2/cyclin A, we discuss the kinases responsible for MCM4 phosphorylation. Phosphorylated MCM4 detected using anti-phospho sera exhibited different affinities for chromatin. Studies on the nuclear localization of chromatin-bound MCM4 phosphorylated at sites 3 and 32 suggested that they are not generally colocalized with replicating DNA. Unexpectedly, MCM4 phosphorylated at site 32 was enriched in the nucleolus through the cell cycle. These results suggest that phosphorylation of MCM4 has several distinct and site-specific roles in the function of MCM during the mammalian cell cycle. 相似文献
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Chromatin association of human origin recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis
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Evidence obtained from studies with yeast and Xenopus indicate that the initiation of DNA replication is a multistep process. The origin recognition complex (ORC), Cdc6p, and minichromosome maintenance (MCM) proteins are required for establishing prereplication complexes, upon which initiation is triggered by the activation of cyclin-dependent kinases and the Dbf4p-dependent kinase Cdc7p. The identification of human homologues of these replication proteins allows investigation of S-phase regulation in mammalian cells. Using centrifugal elutriation of several human cell lines, we demonstrate that whereas human Orc2 (hOrc2p) and hMcm proteins are present throughout the cell cycle, hCdc6p levels vary, being very low in early G(1) and accumulating until cells enter mitosis. hCdc6p can be polyubiquitinated in vivo, and it is stabilized by proteasome inhibitors. Similar to the case for hOrc2p, a significant fraction of hCdc6p is present on chromatin throughout the cell cycle, whereas hMcm proteins alternate between soluble and chromatin-bound forms. Loading of hMcm proteins onto chromatin occurs in late mitosis concomitant with the destruction of cyclin B, indicating that the mitotic kinase activity inhibits prereplication complex formation in human cells. 相似文献
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Site-specific ORC binding,pre-replication complex assembly and DNA synthesis at Schizosaccharomyces pombe replication origins
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Previous studies have shown that the Schizo saccharomyces pombe Orc4 subunit is solely responsible for in vitro binding of origin recognition complex (ORC) to specific AT-rich sites within S.pombe replication origins. Using ARS3001, a S.pombe replication origin consisting of four genetically required sites, we show that, in situ as well as in vitro, Orc4 binds strongly to the Delta3 site, weakly to the Delta6 site and not at all to the remaining sequences. In situ, the footprint over Delta3 is extended during G(1) phase, but only when Cdc18 is present and Mcm proteins are bound to chromatin. Moreover, this footprint extends into the adjacent Delta2 site, where leading strand DNA synthesis begins. Therefore, we conclude that ARS3001 consists of a single primary ORC binding site that assembles a pre-replication complex and initiates DNA synthesis, plus an additional novel origin element (Delta9) that neither binds ORC nor functions as a centromere, but does bind an as yet unidentified protein throughout the cell cycle. Schizosaccharomyces pombe may be an appropriate paradigm for the complex origins found in the metazoa. 相似文献
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Motility of murine lymphocytes during transit through cell cycle. Analysis by a new in vitro assay 总被引:1,自引:0,他引:1
The relationship between the basal (spontaneous) motility of murine lymphocytes and their position in the cell cycle was examined in a new collagen gel motility assay system. Concanavalin A-stimulated or control lymphocytes were allowed to locomote into slabs of type I collagen gel. The assay configuration permitted extraction of both total populations and locomotory subpopulations as viable, single-cell suspensions suitable for phenotypic and cell analysis. Concanavalin A stimulation resulted in a significant increase in the mean distance traveled by the leading cell front in 4 hr, from 23 microns (controls) to 67 microns. The estimated percentage of motile cells increased from 0.9 to 2.8%. Similar increases were observed after 18 hr of locomotion. The SIg+, Thy-1+, L3T4+, and Ly-2+ subsets exhibited equivalent increases in motility. Total populations and locomotory subpopulations were allowed to incorporate 5-bromo-2'-deoxyuridine, and their cell cycle profiles were compared by dual parameter anti-5-bromo-2'-deoxyuridine, propidium iodide fluorescence analysis. Total population and locomotory subpopulations did not differ significantly with respect to the ratio G0/G1:S, indicating that lymphocytes in these two phases exhibited approximately equal motility. Cells in late S and G2 + M were significantly less motile; locomotory subpopulations contained 60 to 75% fewer G2 + M cells than the total populations from which they were derived. Taken together, the results indicate that the concanavalin A-induced increase in motility commences before S phase and that motility diminishes shortly before or during G2 + M. 相似文献
18.
MuB, a protein essential for replicative DNA transposition by the bacteriophage Mu, is an ATPase that assembles into a polymeric complex on DNA. We used total internal reflection fluorescence microscopy to observe the behavior of MuB polymers on single molecules of DNA. We demonstrate that polymer assembly is initiated by a stochastic nucleation event. After nucleation, polymer assembly occurs by a mechanism involving the sequential binding of small units of MuB. MuB that bound to A/T-rich regions of the DNA assembled into large polymeric complexes. In contrast, MuB that bound outside of the A/T-rich regions failed to assemble into large oligomeric complexes. Our data also show that MuB does not catalyze multiple rounds of ATP hydrolysis while remaining bound to DNA. Rather, a single ATP is hydrolyzed, then MuB dissociates from the DNA. Finally, we show that "capping" of the enhanced green fluorescent protein-MuB polymer ends with unlabeled MuB dramatically slows, but does not halt, dissociation. This suggests that MuB dissociation occurs through both an end-dependent mechanism and a slower mechanism wherein subunits dissociate from the polymer interior. 相似文献
19.
Stuermer A Hoehn K Faul T Auth T Brand N Kneissl M Pütter V Grummt F 《European journal of cell biology》2007,86(1):37-50
Initiation of eukaryotic DNA replication is achieved by the sequential binding of different proteins to origins of DNA replication. Using EGFP-tagged initiator proteins and immunofluorescence techniques we found that most of the ORC and the MCM subunits are localised at centrosomes and are colocalised with the polo-like protein kinase, Plk1. Yeast two-hybrid studies revealed interactions of Plk1 with the Mcm2 as well as the Orc2 protein. Co-immunoprecipitations showed an interaction of Plk1 with Mcm2 as well as interactions of gamma-tubulin with Mcm3 and Orc2, respectively. An in vitro phosphorylation assay showed that the Orc2 protein is a substrate of Plk1. Depletion of Orc2 and Mcm3 by siRNA leads to an inhibition of cell proliferation, an altered cell cycle distribution as well as to multinucleated cells with insufficiently organised microtubules. These results indicate an important role of the MCM and ORC proteins in mitosis besides their described role in the establishment of the pre-replicative complex. 相似文献
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Cell cycle modifications are among the early events which take place during the induced differentiation of murine erythroleukemia (MEL) cells; a transient accumulation of the cells in the G1 phase of the cell cycle, followed by a re-entry of the cells into a proliferation state, has been described. In order to characterize a putative role of serum in such variations, we have studied the modifications of the cell cycle parameters when cells were induced to differentiate in the presence or in the absence of seric factors. We show that, in the absence of exogenous factors brought by serum, the G1 accumulation was enhanced both in amplitude and in duration, but cells were still able to bypass the G1 block and re-enter into the S phase. These results indicate that the resumption of cell proliferation after the transient block is under synergistic control of seric and endogenous factors, but these later are sufficient to overcome the block. However, MEL cells were unable to differentiate in the absence of seric factors, as measured by the number of benzidine-positive cells during induction with hexamethylene-bisacetamide (HMBA) or butyric acid. This capacity to differentiate was recovered when serum was added back to the culture medium, and the efficiency of recovery was maximal when cells underwent a full round of DNA replication in the presence of serum after the G1 block. The analysis of two molecular markers of cell differentiation confirmed these results. 相似文献