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1.
Replication licensing--defining the proliferative state? 总被引:16,自引:0,他引:16
The proliferation of eukaryotic cells is a highly regulated process that depends on the precise duplication of chromosomal DNA in each cell cycle. Regulation of the replication licensing system, which promotes the assembly of complexes of proteins termed Mcm2-7 onto replication origins, is responsible for preventing re-replication of DNA in a single cell cycle. Recent work has shown how the licensing system is directly controlled by cyclin-dependent kinases (CDKs). Repression of origin licensing is emerging as a ubiquitous route by which the proliferative capacity of cells is lowered, and Mcm2-Mcm7 proteins show promise as diagnostic markers of early cancer stages. These results have prompted us to propose a functional distinction between the proliferative state and the non-proliferative state (including G0) depending on whether origins are licensed. 相似文献
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After ultraviolet light induction of Escherichia coli K-12 strain W3350(λ), several structural intermediate forms of phage deoxyribonucleic acid (DNA) are synthesized. The early defective lysogens of λ, sus O8, sus P3, and T11, were found to synthesize none of the DNA structural intermediates. A lysogen believed to be defective in all known phage activities, λsus N7, was found to be able to synthesize an early phage DNA intermediate. The lysogen λsus Q21, defective in late phage functions, is able to synthesize the early phage DNA intermediate and a concatenated molecule of greater molecular weight than the mature λ DNA. 相似文献
4.
Epstein–Barr virus (EBV) is a paradigm for human tumor viruses: it is the first virus recognized to cause cancer in people; it causes both lymphomas and carcinomas; yet these tumors arise infrequently given that most people in the world are infected with the virus. EBV is maintained extrachromosomally in infected normal and tumor cells. Eighty-four percent of these viral plasmids replicate each S phase, are licensed, require a single viral protein for their synthesis, and can use two functionally distinct origins of DNA replication, oriP, and Raji ori. Eighty-eight percent of newly synthesized plasmids are segregated faithfully to the daughter cells. Infectious viral particles are not synthesized under these conditions of latent infection. This plasmid replication is consistent with survival of EBV’s host cells. Rare cells in an infected population either spontaneously or following exogenous induction support EBV’s lytic cycle, which is lethal for the cell. In this case, the viral DNA replicates 100-fold or more, uses a third kind of viral origin of DNA replication, oriLyt, and many viral proteins. Here we shall describe the three modes of EBV’s replication as a function of the viral origins used and the viral and cellular proteins that mediate the DNA synthesis from these origins focusing, where practical, on recent advances in our understanding. 相似文献
5.
Can Replication Save Noisy Microarray Data? 总被引:1,自引:0,他引:1
Wernisch L 《Comparative and Functional Genomics》2002,3(4):372-374
6.
Replication Origins and Timing of Temporal Replication in Budding Yeast: How to Solve the Conundrum?
Similarly to metazoans, the budding yeast Saccharomyces cereviasiae replicates its genome with a defined timing. In this organism, well-defined, site-specific origins, are efficient and fire in almost every round of DNA replication. However, this strategy is neither conserved in the fission yeast Saccharomyces pombe, nor in Xenopus or Drosophila embryos, nor in higher eukaryotes, in which DNA replication initiates asynchronously throughout S phase at random sites. Temporal and spatial controls can contribute to the timing of replication such as Cdk activity, origin localization, epigenetic status or gene expression. However, a debate is going on to answer the question how individual origins are selected to fire in budding yeast. Two opposing theories were proposed: the “replicon paradigm” or “temporal program” vs. the “stochastic firing”. Recent data support the temporal regulation of origin activation, clustering origins into temporal blocks of early and late replication. Contrarily, strong evidences suggest that stochastic processes acting on origins can generate the observed kinetics of replication without requiring a temporal order. In mammalian cells, a spatiotemporal model that accounts for a partially deterministic and partially stochastic order of DNA replication has been proposed. Is this strategy the solution to reconcile the conundrum of having both organized replication timing and stochastic origin firing also for budding yeast? In this review we discuss this possibility in the light of our recent study on the origin activation, suggesting that there might be a stochastic component in the temporal activation of the replication origins, especially under perturbed conditions. 相似文献
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The replicon model has initiated a major research line in molecular biology: the study of DNA replication mechanisms. Until now, the majority of studies have focused on a limited set of model organisms, mainly from Bacteria or Opisthokont eukaryotes (human, yeasts) and a few viral systems. However, molecular evolutionists have shown that the living world is more complex and diverse than believed when the operon model was proposed. Comparison of DNA replication proteins in the three domains, Archaea, Bacteria, and Eukarya, have surprisingly revealed the existence of two distinct sets of non-homologous cellular DNA replication proteins, one in Bacteria and the other in Archaea and Eukarya, suggesting that the last universal common ancestor possibly still had an RNA genome. A major puzzle is the presence in eukaryotes of the unfaithful DNA polymerase alpha (Pol α) to prime Okazaki fragments. Interestingly, Pol α is specifically involved in telomere biosynthesis, and its absence in Archaea correlates with the absence of telomeres. The recent discovery of telomere-like GC quartets in eukaryotic replication origins suggests a link between Pol α and the overall organization of the eukaryotic chromosome. As previously proposed by Takemura, Pol α might have originated from a mobile element of viral origin that played a critical role in the emergence of the complex eukaryotic genomes. Notably, most large DNA viruses encode DNA replication proteins very divergent from their cellular counterparts. The diversity of viral replication machineries compared to cellular ones suggests that DNA and DNA replication mechanisms first originated and diversified in the ancient virosphere, possibly explaining why they are so many different types of replication machinerie. 相似文献
11.
L Zhang S Yin W Tan D Xiao Y Weng W Wang T Li J Shi L Shuai H Li J Zhou JP Allain C Li 《PloS one》2012,7(8):e42455
Recombinant interferon-γ (IFNγ) production in cultured lentivirus (LV) was explored for inhibition of target virus in cells co-infected with adenovirus type 5 (Ad5). The ability of three different promoters of CMV, EF1α and Ubiquitin initiating the enhanced green fluorescence protein (GFP) activities within lentiviruses was systematically assessed in various cell lines, which showed that certain cell lines selected the most favorable promoter driving a high level of transgenic expression. Recombinant IFNγ lentivirus carrying CMV promoter (LV-CMV-IFNγ) was generated to co-infect 293A cells with a viral surrogate of recombinant GFP Ad5 in parallel with LV-CMV-GFP control. The best morphologic conditions were observed from the two lentiviruses co-infected cells, while single adenovirus infected cells underwent clear pathologic changes. Viral load of adenoviruses from LV-CMV-IFNγ or LV-CMV-GFP co-infected cell cultures was significantly lower than that from adenovirus alone infected cells (P = 0.005–0.041), and the reduction of adenoviral load in the co-infected cells was 86% and 61%, respectively. Ad5 viral load from LV-CMV-IFNγ co-infected cells was significantly lower than that from LV-CMV-GFP co-infection (P = 0.032), which suggested that IFNγ rather than GFP could further enhance the inhibition of Ad5 replication in the recombinant lentivirus co-infected cells. The results suggest that LV-CMV-IFNγ co-infection could significantly inhibit the target virus replication and might be a potential approach for alternative therapy of severe viral diseases. 相似文献
12.
DePamphilis ML 《BioEssays : news and reviews in molecular, cellular and developmental biology》1999,21(1):5-16
The process by which eukaryotic cells decide when and where to initiate DNA replication has been illuminated in yeast, where specific DNA sequences (replication origins) bind a unique group of proteins (origin recognition complex) next to an easily unwound DNA sequence at which replication can begin. The origin recognition complex provides a platform on which additional proteins assemble to form a pre-replication complex that can be activated at S-phase by specific protein kinases. Remarkably, multicellular eukaryotes, such as frogs, flies, and mammals (metazoa), have counterparts to these yeast proteins that are required for DNA replication. Therefore, one might expect metazoan chromosomes to contain specific replication origins as well, a hypothesis that has long been controversial. In fact, recent results strongly support the view that DNA replication origins in metazoan chromosomes consist of one or more high frequency initiation sites and perhaps several low frequency ones that together can appear as a nonspecific initiation zone. Specific replication origins are established during G1-phase of each cell cycle by multiple parameters that include nuclear structure, chromatin structure, DNA sequence, and perhaps DNA modification. Such complexity endows metazoa with the flexibility to change both the number and locations of replication origins in response to the demands of animal development. 相似文献
13.
Bielinsky AK 《Cell cycle (Georgetown, Tex.)》2003,2(4):307-309
During the G1 phase of the cell cycle, replication origins are prepared to fire, a process that is referred to as origin licensing. It was often pondered what a cell's fate would be if not all of its replication origins were licensed and subsequently activated during S phase. One obvious prediction was that S phase would simply be prolonged. As it turns out, however, the consequences are much more complex. A short G1 phase enforced by premature entry into S phase, or other events that negatively affect origin licensing, will ultimately compromise the cell's ability to complete DNA replication before entering mitosis. As a result, the cell becomes genomically unstable when it attempts to repair unreplicated DNA during anaphase. Thus, the density of active replication origins in the chromosomes of eukaryotic cells determines S phase dynamics and chromosome stability during mitosis. 相似文献
14.
Darren E. Casteel Shunhui Zhuang Ying Zeng Fred W. Perrino Gerry R. Boss Mehran Goulian Renate B. Pilz 《The Journal of biological chemistry》2009,284(9):5807-5818
α-Accessory factor (AAF) stimulates the activity of DNA
polymerase-α·primase, the only enzyme known to initiate DNA
replication in eukaryotic cells (Goulian, M., Heard, C. J.,
and Grimm, S. L. (1990) J. Biol. Chem. 265
,13221
-13230). We purified the AAF
heterodimer composed of 44- and 132-kDa subunits from cultured cells and
identified full-length cDNA clones using amino acid sequences from internal
peptides. AAF-132 demonstrated no homologies to known proteins; AAF-44,
however, is evolutionarily related to the 32-kDa subunit of replication
protein A (RPA-32) and contains an oligonucleotide/oligosaccharide-binding
(OB) fold domain similar to the OB fold domains of RPA involved in
single-stranded DNA binding. Epitope-tagged versions of AAF-44 and -132 formed
a complex in intact cells, and purified recombinant AAF-44 bound to
single-stranded DNA and stimulated DNA primase activity only in the presence
of AAF-132. Mutations in conserved residues within the OB fold of AAF-44
reduced DNA binding activity of the AAF-44·AAF-132 complex.
Immunofluorescence staining of AAF-44 and AAF-132 in S phase-enriched HeLa
cells demonstrated punctate nuclear staining, and AAF co-localized with
proliferating cell nuclear antigen, a marker for replication foci containing
DNA polymerase-α·primase and RPA. Small interfering RNA-mediated
depletion of AAF-44 in tumor cell lines inhibited
[methyl-3H]thymidine uptake into DNA but did not affect
cell viability. We conclude that AAF shares structural and functional
similarities with RPA-32 and regulates DNA replication, consistent with its
ability to increase polymerase-α·primase template affinity and
stimulate both DNA primase and polymerase-α activities in
vitro.In eukaryotic cells, DNA replication is initiated at multiple origins
internal to each chromosome; the origin recognition complex recruits cell
division cycle and minichromosome maintenance proteins to form a preinitiation
complex (1). At the
G1-S phase transition, the latter complex is activated by
cyclin-dependent protein kinases leading to formation of an initiation complex
that alters local DNA structure through DNA helicase activity
(1,
2). The replication protein A
(RPA)2 is recruited to
bind and stabilize single-stranded DNA (ssDNA) produced by the initiation
complex (3,
4). RPA serves as an auxiliary
factor for DNA polymerase-α (pol-α)·primase: it stabilizes
the protein complex by direct interaction with both pol-α and primase
subunits, and it reduces the misincorporation rate of pol-α, acting as a
“fidelity clamp”
(5,
6). The
pol-α·primase complex consists of four subunits, including the
catalytic pol-α subunit (p185), a regulatory B subunit (p70), and two
primase subunits (p49 and p58). On an ssDNA template, the primase synthesizes
short RNA primers from ribonucleoside triphosphates (rNTPs), which are
elongated by pol-α in the presence of deoxyribonucleoside triphosphates
(dNTPs) to form short DNA fragments. Through mechanisms requiring other
replication factors, pol-α·primase is replaced by the more
processive DNA polymerases pol-δ and pol-ε
(7). Pol-ε synthesizes the
leading strand, whereas pol-δ completes each Okazaki fragment initiated
by pol-α·primase on the lagging strand and proofreads errors made
by pol-α (7). The
initiator RNA and DNA fragments are later removed by nucleases, and the
Okazaki fragments are sealed by DNA ligase
(7).The pol-α·primase complex is the only eukaryotic DNA
polymerase able to initiate DNA synthesis de novo. In addition to
initiating DNA replication and synthesizing Okazaki fragments, it appears to
be one of the final targets of cell cycle checkpoint pathways that couple DNA
replication to DNA damage response
(2,
8). The role of RPA in
initiation, elongation, and completion of lagging strand DNA synthesis has
been thoroughly investigated
(3,
9), but in vitro
studies suggest that some additional factors that promote the rapidity of DNA
replication in vivo are still lacking
(2).In the course of purifying pol-α·primase from extracts of
cultured mouse L1210 cells, we identified a factor we named α-accessory
factor (AAF) that stimulates pol-α·primase activity in
vitro (10,
11). The protein has a native
molecular mass of ∼150 kDa as determined from its sedimentation
coefficient and Stokes radius and is composed of two subunits of ∼132 and
∼44 kDa. AAF stimulates pol-α·primase activity with several
different templates and types of reactions: (i) It stimulates selfprimed
reactions with poly(dT), poly(dI·dT), or single-stranded circular DNA;
(ii) it stimulates primed reactions with poly(dA)·oligo(dT) and
multiply primed DNA in the absence of rNTPs, indicating that it affects
pol-α activity when no primers are being made; and (iii) it stimulates
primase activity on ssDNA in the absence of dNTPs, showing that it can enhance
RNA primer synthesis in the absence of DNA synthesis
(11). AAF increases the
template affinity and processivity of pol-α·primase
(12). AAF is highly specific
for pol-α·primase and has no effect on the other mammalian DNA
polymerases β, γ, or δ or on the DNA
polymerase·primase complexes from Drosophila and
Saccharomyces cerevisiae
(11).The cloning of both AAF subunits based on peptide sequences obtained from
the purified protein allowed us now to further characterize the
AAF-44·AAF-132 complex structurally and functionally. Based on siRNA
experiments in cancer cell lines, AAF appears to regulate DNA replication
in vivo. 相似文献
15.
《Cell cycle (Georgetown, Tex.)》2013,12(4):306-308
During the G1 phase of the cell cycle, replication origins are prepared to fire, a process that is referred to as origin licensing. It was often pondered what a cell’s fate would be if not all of its replication origins were licensed and subsequently activated during S phase. One obvious prediction was that S phase would simply be prolonged. As it turns out, however, the consequences are much more complex. A short G1 phase enforced by premature entry into S phase, or other events that negatively affect origin licensing, will ultimately compromise the cell’s ability to complete DNA replication before entering mitosis. As a result, the cell becomes genomically unstable when it attempts to repair unreplicated DNA during anaphase. Thus, the density of active replication origins in the chromosomes of eukaryotic cells determines S phase dynamics and chromosome stability during mitosis. 相似文献
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Jinyang Zhang Xiaopeng Wu Jie Zan Yongping Wu Chengjin Ye Xizhen Ruan Jiyong Zhou 《Journal of virology》2013,87(13):7608-7621
Rabies, as the oldest known infectious disease, remains a serious threat to public health worldwide. The eukaryotic cytosolic chaperonin TRiC/CCT complex facilitates the folding of proteins through ATP hydrolysis. Here, we investigated the expression, cellular localization, and function of neuronal CCTγ during neurotropic rabies virus (RABV) infection using mouse N2a cells as a model. Following RABV infection, 24 altered proteins were identified by using two-dimensional electrophoresis and mass spectrometry, including 20 upregulated proteins and 4 downregulated proteins. In mouse N2a cells infected with RABV or cotransfected with RABV genes encoding nucleoprotein (N) and phosphoprotein (P), confocal microscopy demonstrated that upregulated cellular CCTγ was colocalized with viral proteins N and P, which formed a hollow cricoid inclusion within the region around the nucleus. These inclusions, which correspond to Negri bodies (NBs), did not form in mouse N2a cells only expressing the viral protein N or P. Knockdown of CCTγ by lentivirus-mediated RNA interference led to significant inhibition of RABV replication. These results demonstrate that the complex consisting of viral proteins N and P recruits CCTγ to NBs and identify the chaperonin CCTγ as a host factor that facilitates intracellular RABV replication. This work illustrates how viruses can utilize cellular chaperonins and compartmentalization for their own benefit. 相似文献
18.
Yong-Jun Liu Christopher J. Papasian Jian-Feng Liu James Hamilton Hong-Wen Deng 《PloS one》2008,3(12)
With the advent of genome-wide association (GWA) studies, researchers are hoping that reliable genetic association of common human complex diseases/traits can be detected. Currently, there is an increasing enthusiasm about GWA and a number of GWA studies have been published. In the field a common practice is that replication should be used as the gold standard to validate an association finding. In this article, based on empirical and theoretical data, we emphasize that replication of GWA findings can be quite difficult, and should not always be expected, even when true variants are identified. The probability of replication becomes smaller with the increasing number of independent GWA studies if the power of individual replication studies is less than 100% (which is usually the case), and even a finding that is replicated may not necessarily be true. We argue that the field may have unreasonably high expectations on success of replication. We also wish to raise the question whether it is sufficient or necessary to treat replication as the ultimate and gold standard for defining true variants. We finally discuss the usefulness of integrating evidence from multiple levels/sources such as genetic epidemiological studies (at the DNA level), gene expression studies (at the RNA level), proteomics (at the protein level), and follow-up molecular and cellular studies for eventual validation and illumination of the functional relevance of the genes uncovered. 相似文献
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