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Results for “Replicability”

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At least 217 records · Page 12Linked to original sources

PubMed1992

Replication of nuclear and mitochondrial DNA in X-ray-damaged cells: evidence for a nuclear-specific mechanism that down-regulates replication.

The mechanism by which X rays inhibit DNA replication has been investigated in three distinct populations of DNA molecules in human cells: (a) large chromosomal DNA, (b) a population of 50-100 10.3-kb nuclear episomal plasmids per cell, and (c) a population of about 500 16-kb cytoplasmic mitochondrial DNA molecules per cell. DNA replication was inhibited by X rays in nuclear chromosomal and plasmid DNA, but not in mitochondrial DNA. The mechanism by which ionizing radiation inhibits DNA replication must therefore be nuclear-specific and is unlikely to involve diffusible low-molecular-weight substances. Since mitochondrial DNA exists in the cell as independent 16-kb circular molecules and responds to radiation as would be expected for small targets, the implication for nuclear plasmids is that their replication is regulated by a large target. A current model for DNA replication involves the movement of DNA through replication centers made up of polymerases, helicases, and associated replication enzymes that are attached to a matrix. The difference in the response to X rays between mitochondrial DNA and nuclear plasmid DNA can be explained if nuclear plasmids are tightly associated with chromosomal DNA and attached to the matrix, and are coordinately replicated.

DNA Replication↗

PubMed2004

[In vitro gene therapy of hepatocellular carcinoma using replication-defective and tumor-specific replication-competent adenovirus carrying interleukin-12 gene].

OBJECTIVE: To evaluate the therapeutic effect and the expression level of a tumor-specific replication-competent adenovirus and a replication-defective adenovirus expression mouse recombinant IL-12 (mIL-12) gene on hepatocellular carcinoma (HCC) in vitro. METHODS: The cytotoxicity of replication-competent adenovirus with E1B-55 000 attenuated CNHK200-mIL12 and ONYX-015 (dl1520), and replication-defective adenovirus Adv-mIL12 were evaluated by MTT and replication assay in two HCC cell lines (HepG2 and Hep3B) and human normal hepatocyte line (LO2). Western blot and ELISA were used to determine the expression level of mIL-12. RESULTS: CNHK200-mIL12 replicated in HepG2 and Hep3B with an increase of 3,160-fold and 630-fold respectively in 96 h post-infection. CNHK200-mIL12 could kill HepG2 and Hep3B cells at a very low MOI (Multiplicity of Infection) and in short time course (HepG2:MOI = 0.2, on day 4; Hep3B:MOI = 0.005, on day 2), while it had no significant effect on LO2. Furthermore, the expressing level of mIL-12 in CNHK200-mIL12 treated HCC cell lines was much higher than that in Adv-mIL12 treated one (HepG2 101-fold, Hep3B 20-fold respectively). CONCLUSION: Replication-competent adenovirus is more effective than replication-defective adenovirus in both cytotoxicity and efficiency of gene transfer in HCC, and holds great promise in the area of HCC therapy.

Adenoviridae↗

PubMed1990

Host virus interactions in the initiation of bacteriophage lambda DNA replication. Recruitment of Escherichia coli DnaB helicase by lambda P replication protein.

The bacteriophage lambda P protein promoters replication of the phage chromosome by recruiting a key component of the cellular replication machinery to the viral origin. Specifically, P protein delivers one or more molecules of Escherichia coli DnaB helicase to a nucleoprotein structure formed by the lambda O initiator at the lambda replication origin. Using purified proteins, we have examined the features of the pivotal host virus interaction between P and DnaB. These two proteins interact in vitro to form a P.DnaB protein complex that can be resolved by sedimentation or by chromatography on DEAE-cellulose from the individual free proteins. The sedimentation coefficient of the P.DnaB complex, 13 S, suggests a size larger than that of free DnaB hexamer (Mr = 313,600). The P.DnaB complex isolated by glycerol gradient sedimentation contains approximately three protomers of P/DnaB hexamer, consistent with a molecular weight of 393,000. The isolated P.DnaB complex functions in vitro in the initiation of lambda DNA replication. Interaction of P with DnaB strongly suppressed both the intrinsic DNA-dependent ATPase activity of DnaB, as well as the capacity of DnaB to assist E. coli primase in the general priming reaction. Formation of a P.DnaB protein complex also blocked DnaB from functioning in the initiation of E. coli DNA replication in vitro. The physical and functional properties of lambda P protein suggest that it is a viral analogue of the E. coli DnaC replication protein. Like P, DnaC also binds to DnaB (Wickner, S., and Hurwitz, J. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 921-925), but unlike P, DnaC stimulates DnaB-mediated general priming. When viral P and bacterial DnaC replication proteins were placed in direct competition with one another for binding to DnaB, the viral protein was clearly predominant. For example, a 5-fold molar excess of DnaC protein only partially reversed the inhibitory effect of P on general priming. Furthermore, when a preformed DnaC.DnaB protein complex was incubated briefly with P protein, it was readily converted into a P.DnaB protein complex and the bulk of the bound DnaC was released as free protein. It is likely that the capacity of the lambda P protein to outcompete the analogous host protein for binding to the bacterial DnaB helicase is the critical molecular event enabling infecting phage to recruit cellular replication proteins required for initiation of DNA synthesis at the viral origin.

Bacterial Proteins↗

PubMedDate not supplied

Quantitative and in vivo activity of adenoviral-producing cells made by cotransduction of a replication-defective adenovirus and a replication-enabling plasmid.

Achieving limited recombinant viral replication may provide a means of amplifying viral-mediated gene transfer in vivo. We have previously shown that cotransduction of an E1-defective adenovirus with a plasmid containing the deleted E1 functions into prostate carcinoma cells resulted in E1-defective virus production by those cells. The studies described here have extended these findings to more firmly establish the capacity of the trans complementation approach to achieve amplification of recombinant viral transgene expression. The recombinant virus used for all the studies was AdCMV-luc which contained a luciferase expression cassette; the replication-enabling plasmid, pE1, encoded the E1 functions deleted from AdCMV-luc. Quantitative in vitro studies with the HeLa cell line showed that for each plaque forming unit of AdCMV-luc originally exposed to the cells, 0.54 x 10(3) new replication-defective viruses were detected in supernatants and lysates over the following 4 days. Multiple cell lines were shown to support new virus production following cotransduction of AdCMV-luc and pE1. Small numbers of replication-competent viruses were detected in the lysates and supernatants from the cotransduced cells such that for every 10(5) replication-defective viruses approximately two replication-competent viruses were produced. Tumor nodules produced by engrafting a mixture of AdCMV-luc/pE1-cotransduced HeLa cells with uninfected HeLa cells yielded much higher levels of luciferase expression than control tumors containing mixtures of cells infected with AdCMV-luc alone. In total, these results demonstrate new virus production by cells receiving a replication-defective adenovirus and a replication-enabling plasmid are capable of amplifying recombinant viral transgene expression in vivo.

Adenoviridae↗

PubMed1996

Mechanisms restricting DNA replication to once per cell cycle: MCMS, pre-replicative complexes and kinases.

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.

Journal Article↗

PubMed1980

Plasmid replication functions: two distinct segments of plasmid R1, RepA and RepD, express incompatibility and are capable of autonomous replication.

The genetic determinants for replication and incompatibility of plasmid R1 were investigated by gene cloning methods, and three types of R1 miniplasmid derivatives were generated. The first, exemplified by plasmid pKT300, consisted of a single BglII endonuclease-generated deoxyribonucleic acid fragment derived from the R1 region that is located between the determinants for conjugal transfer and antibiotic resistance. Two types of miniplasmids could be formed from PstI endonuclease-generated fragments of pKT300. One of these, which is equivalent to miniplasmids previously generated from plasmids R1-19 and R1-19B2, consisted of two adjacent PstI fragments that encode the RepA replication system of plasmid R1. The other type contained a segment of R1, designated the RepD replication region, that is adjacent to the RepA region and that has not been identified previously as having the capacity for autonomous replication. Plasmid R1, therefore, contained two distinct deoxyribonucleic acid segments capable of autonomous replication. The RepA-RepD miniplasmid pKT300 had a copy number about eightfold higher than that of R1 and hence lacked a determinant for the regulation of plasmid copy number. Like R1, it was maintained stably in dividing bacteria. RepA miniplasmids had copy numbers which were two- to fourfold higher than that of R1 (i.e., which were lower than that of pKT300) and were maintained slightly less stably than those of pKT300 and R1. The RepD miniplasmid was not maintained stably in dividing bacteria. Previous experiments have shown that incompatibility of IncFII group plasmids is specified by a plasmid copy control gene. Despite the fact that RepA miniplasmids of R1 were defective in copy control, they nevertheless expressed incompatibility. This suggests that two genes are responsible for plasmid copy control, one that specifies incompatibility and is located on RepA miniplasmids and another that is located outside of, but adjacent to, the RepA replication region. Hybrid plasmids composed of pBR322 and one PstI fragment from the RepA region, P-8, exhibited incompatibility towards R2 and RepA miniplasmids but not the RepD miniplasmid, whereas hybrids composed of pBR322 and the PstI fragment of the RepD region, P-3, exhibited incompatibility towards R1 and the RepD miniplasmid but not RepA miniplasmids. These results indicate that the two replication systems are functionally distinct and that, although the RepA system is the principal replication system of R1, the RepD system also plays a role in the maintenance of this plasmid.

Cloning, Molecular↗

PubMed1993

SV40 DNA replication intermediates: analysis of drugs which target mammalian DNA replication.

The simian virus 40 chromosome, a model for the mammalian replicon, is a uniquely powerful system for the study of drugs and treatments which target enzymes of the mammalian replication apparatus. High resolution gel electrophoretic analysis of normal and aberrant viral replication intermediates can be used effectively to understand the molecular events of replication failure. These events include breakage of replication forks, aberrant topoisomerase action, failure to separate daughter chromosomes, protein-DNA crosslinking, single and double strand DNA breakage, alterations in topology and inactivation of replication intermediates. The SV40 replication system can also be used to study the recombinational events which often follow drug-induced replication failure.

Animals↗

PubMed2002

Interaction of geminivirus Rep protein with replication factor C and its potential role during geminivirus DNA replication.

Geminivirus DNA replication during the rolling-circle stage depends on the use of a DNA primer, a strategy poorly understood as compared with other eukaryotic viral systems that rely on RNA or protein as primers. Here we have used wheat dwarf virus (WDV) with the aim of elucidating the events leading to recruitment of cell factors at the replication origin. We have identified a novel interaction of WDV Rep, the replication initiation protein, with the large subunit of the wheat replication factor C complex (TmRFC-1). In other systems, the heteropentameric RFC clamp loader complex stimulates loading of DNA polymerase delta to the primer-template. Expression of TmRFC-1 is subjected to cell-cycle regulation, with a peak in early S-phase. We show that WDV Rep stimulates binding of recombinant TmRFC-1 to a model substrate containing a 3'-OH terminus and a WDV Rep-binding site. This was confirmed using cellular fractions enriched for wheat RFC complex, supporting the idea that, in addition to generating a 3'-OH terminus during initiation of DNA replication, WDV Rep could participate in the recruitment of RFC to the newly formed primer. We propose that this pathway may represent an initial event to facilitate the assembly of other replication factors, e.g., PCNA and/or DNA polymerase delta, a model that could also apply to other eukaryotic replicons, such as nanoviruses, circoviruses, and parvoviruses with a similar DNA replication strategy.

Amino Acid Sequence↗

PubMed2001

A DNA replication origin and a replication fork barrier used in vivo in the circular plasmid pKD1.

As in other yeasts, ARS-containing plasmids can be maintained extrachromosomally in Kluyveromyces lactis. Although some fragments of K. lactis DNA have ARS activity in both K. lactis and Saccharomyces cerevisiae, it appears that the sequences required for ARS activity in the two yeasts are different. As an approach to a better understanding of ARS structure and function in K. lactis, we analyzed the replication of the circular plasmid pKD1. We identified a 159-bp sequence able to promote autonomous replication of pKD1 in both yeasts; this fragments contains both a sequence related to the S. cerevisiae ARS consensus sequence and a region of 53% identity to the 40-bp sequence essential for K. lactis KARS101 function. By the analysis of in vivo replication intermediates we provide the first direct evidence that DNA replication initiates at or near the K. lactis ARS element. Replication terminates at the cisacting stability locus of pKD1, which functions as a replication fork barrier (RFB) and is necessary for proper plasmid segregation. RFB activity requires the pKDI gene products that are important for plasmid segregation, suggesting a link between DNA replication termination and plasmid segregation in a eukaryotic organism.

Base Sequence↗

PubMed2006

Interactions of the cellular CCAAT displacement protein and human papillomavirus E2 protein with the viral origin of replication can regulate DNA replication.

Previously, we and others have shown that CCAAT displacement protein (CDP) negatively regulates the papillomavirus promoters. Overexpression of CDP has been shown to inhibit high-risk human papillomavirus virus (HPV) and bovine papillomavirus DNA replication in vivo presumably through reduction in expression of viral replication proteins, E1 and E2. Sequence analysis of the HPV origin indicates several potential CDP-binding sites with one site overlapping the E1-binding site. Therefore, CDP could also negatively regulate papillomavirus replication directly by preventing the loading of the initiation complex. We show here that purified CDP inhibits in vitro HPV DNA replication. Footprint analysis demonstrated that CDP binds the E1-binding site and the TATA box, and that the binding of purified CDP to the E1-binding site is decreased by the addition of purified E2 protein. Consistent with this, E2-independent in vitro HPV replication is inhibited by CDP to a greater extent than E2-dependent replication. These results suggest that binding of E2 at the E2-binding site may play an important role in overcoming the inhibition of E1 initiation complex formation caused by the binding of negative regulators like CDP to the origin of replication.

Base Sequence↗

PubMed2001

The cellular response to DNA damage induced by the enediynes C-1027 and neocarzinostatin includes hyperphosphorylation and increased nuclear retention of replication protein a (RPA) and trans inhibition of DNA replication.

This study examined the cellular response to DNA damage induced by antitumor enediynes C-1027 and neocarzinostatin. Treatment of cells with either agent induced hyperphosphorylation of RPA32, the middle subunit of replication protein A, and increased nuclear retention of RPA. Nearly all of the RPA32 that was not readily extractable from the nucleus was hyperphosphorylated, compared to < or =50% of the soluble RPA. Enediyne concentrations that induced RPA32 hyperphosphorylation also decreased cell-free SV40 DNA replication competence in extracts of treated cells. This decrease did not result from damage to the DNA template, indicating trans-acting inhibition of DNA replication. Enediyne-induced RPA hyperphosphorylation was unaffected by the replication elongation inhibitor aphidicolin, suggesting that the cellular response to enediyne DNA damage was not dependent on elongation of replicating DNA. Neither recovery of replication competence nor reversal of RPA effects occurred when treated cells were further incubated in the absence of drug. C-1027 and neocarzinostatin doses that caused similar levels of DNA damage resulted in equivalent increases in RPA32 hyperphosphorylation and RPA nuclear retention and decreases in replication activity, suggesting a common response to enediyne-induced DNA damage. By contrast, DNA damage induced by C-1027 was at least 5-fold more cytotoxic than that induced by neocarzinostatin.

Aminoglycosides↗

PubMed1985

Initiation of DNA replication on single-stranded DNA templates catalyzed by purified replication proteins of bacteriophage lambda and Escherichia coli.

Initiation of bacteriophage lambda DNA replication at the chromosomal origin depends on the lambda O and P replication proteins. These two viral initiators, together with an Escherichia coli protein fraction, promote the replication in vitro of single-stranded circular DNA chromosomes such as that of bacteriophage M13. This nonspecific strand initiation reaction, which we have termed the "lambda single-strand replication reaction," has now been established with eight purified proteins, each of which is also required for replication of the phage lambda chromosome in vivo. An early rate-limiting step in the overall reaction is the ATP-dependent assembly of an activated nucleoprotein prepriming complex. In this step the lambda O and P initiators cooperate with the E. coli dnaJ and dnaK proteins to transfer the bacterial dnaB protein onto M13 DNA that is coated with the single-stranded DNA-binding protein. Multiple RNA primers are synthesized on each DNA circle when isolated prepriming complex is incubated with primase and rNTPs. In the complete system, DNA polymerase III holoenzyme extends the first primer synthesized into full-length complementary strands. Because the properties of this system are closely analogous to those found for the replication of phi X174 viral DNA by E. coli proteins, we infer that a mobile prepriming or priming complex (primosome) operates in the lambda single-strand replication reaction.

Bacterial Proteins↗

PubMed1989

Purification of replication protein C, a cellular protein involved in the initial stages of simian virus 40 DNA replication in vitro.

The replication of simian virus 40 (SV40) DNA is dependent upon a single viral protein [tumor (T) antigen] and multiple cellular proteins. To define the required cellular proteins, we have made use of a cell-free system that supports the replication of plasmid DNA molecules containing the SV40 origin of replication. We report here the purification from HeLa cell extracts of replication protein C (RP-C), a previously undescribed protein that is required to reconstitute efficient DNA replication in vitro. Highly purified preparations of RP-C contain two closely related polypeptides of 32 and 34 kDa. Preincubation of purified RP-C with T antigen and the DNA template largely eliminates the delay normally observed before the onset of rapid DNA synthesis. In addition, RP-C stimulates the unwinding of duplex DNA molecules containing the SV40 replication origin in a reaction that requires T antigen and a single-stranded DNA binding protein. These observations suggest that RP-C is involved in the initial steps of SV40 DNA replication in vitro.

Antigens, Polyomavirus Transforming↗

PubMed2000

Adozelesin triggers DNA damage response pathways and arrests SV40 DNA replication through replication protein A inactivation.

The cyclopropylpyrroloindole anti-cancer drug, adozelesin, binds to and alkylates DNA. Treatment of human cells with low levels of adozelesin results in potent inhibition of both cellular and simian virus 40 (SV40) DNA replication. Extracts were prepared from adozelesin-treated cells and shown to be deficient in their ability to support SV40 DNA replication in vitro. This effect on in vitro DNA replication was dependent on both the concentration of adozelesin used and the time of treatment but was not due to the presence of adozelesin in the in vitro assay. Adozelesin treatment of cells was shown to result in the following: induction of p53 protein levels, hyperphosphorylation of replication protein A (RPA), and disruption of the p53-RPA complex (but not disruption of the RPA-cdc2 complex), indicating that adozelesin treatment triggers cellular DNA damage response pathways. Interestingly, in vitro DNA replication could be rescued in extracts from adozelesin-treated cells by the addition of exogenous RPA. Therefore, whereas adozelesin and other anti-cancer therapeutics trigger common DNA damage response markers, adozelesin causes DNA replication arrest through a unique mechanism. The S phase checkpoint response triggered by adozelesin acts by inactivating RPA in some function essential for SV40 DNA replication.

Antineoplastic Agents, Alkylating↗

PubMed1992

Identification of nuclear pre-replication centers poised for DNA synthesis in Xenopus egg extracts: immunolocalization study of replication protein A.

We demonstrate by immunofluorescence that a 70-kD protein (P70) purified from Xenopus egg extracts is associated with subnuclear foci (about 200) which we propose to be an assembly of DNA pre-replication centers (preRCs). A cDNA encoding this protein reveals that P70 is the Xenopus homologue of replication protein A (RPA also called RF-A). RPA is know to be a cellular, three-subunit single-stranded DNA binding protein, which assists T-antigen in the assembly of the pre-priming complex in the SV40 replication system. The punctated preRCs exist transiently; they form post-mitotically during the period of nuclear membrane breakdown and disappear during ongoing DNA replication. P70 is homogeneously associated with chromatin at the later stages of the S-phase and is displaced from chromatin post replication, so that P70 cannot be detected on mitotic chromosomes. Double-immunofluorescence studies using biotin-dUTP demonstrate that initiation of DNA synthesis is confined to preRCs, resulting in the punctated replication pattern observed previously by others. PreRCs form efficiently on decondensed chromatin in membrane-free egg extracts if ATP and divalent cations are present. Our results suggest that preRCs are composed of an assembly of a large number of pre-initiation replication complexes poised for initiation at discreet subnuclear regions prior to nuclear reconstruction and initiation of DNA synthesis.

Amino Acid Sequence↗

PubMed2001

Activation of mammalian Chk1 during DNA replication arrest: a role for Chk1 in the intra-S phase checkpoint monitoring replication origin firing.

Checkpoints maintain order and fidelity in the cell cycle by blocking late-occurring events when earlier events are improperly executed. Here we describe evidence for the participation of Chk1 in an intra-S phase checkpoint in mammalian cells. We show that both Chk1 and Chk2 are phosphorylated and activated in a caffeine-sensitive signaling pathway during S phase, but only in response to replication blocks, not during normal S phase progression. Replication block-induced activation of Chk1 and Chk2 occurs normally in ataxia telangiectasia (AT) cells, which are deficient in the S phase response to ionizing radiation (IR). Resumption of synthesis after removal of replication blocks correlates with the inactivation of Chk1 but not Chk2. Using a selective small molecule inhibitor, cells lacking Chk1 function show a progressive change in the global pattern of replication origin firing in the absence of any DNA replication. Thus, Chk1 is apparently necessary for an intra-S phase checkpoint, ensuring that activation of late replication origins is blocked and arrested replication fork integrity is maintained when DNA synthesis is inhibited.

Alkaloids↗

PubMed2006

Excess Mcm2-7 license dormant origins of replication that can be used under conditions of replicative stress.

In late mitosis and early G1, replication origins are licensed for subsequent use by loading complexes of the minichromosome maintenance proteins 2-7 (Mcm2-7). The number of Mcm2-7 complexes loaded onto DNA greatly exceeds the number of replication origins used during S phase, but the function of the excess Mcm2-7 is unknown. Using Xenopus laevis egg extracts, we show that these excess Mcm2-7 complexes license additional dormant origins that do not fire during unperturbed S phases because of suppression by a caffeine-sensitive checkpoint pathway. Use of these additional origins can allow complete genome replication in the presence of replication inhibitors. These results suggest that metazoan replication origins are actually comprised of several candidate origins, most of which normally remain dormant unless cells experience replicative stress. Consistent with this model, using Caenorhabditis elegans, we show that partial RNAi-based knockdown of MCMs that has no observable effect under normal conditions causes lethality upon treatment with low, otherwise nontoxic, levels of the replication inhibitor hydroxyurea.

Adenosine Triphosphatases↗

PubMed1995

Origin of replication of the Bacillus subtilis chromosome: in vitro approach to the isolation of early replicating segments.

We have developed a permeable cell system for the study of the molecular mechanisms involved in the control and initiation of DNA replication at the origin of the Bacillus subtilis chromosome. Our system take advantage of the synchronous initiation of DNA replication that occurs in outgrowing B. subtilis spores and the curtailment of DNA elongation by novobiocin. Early replicating DNA sequences were identified by the use of 5-mercury-dCTP as substrate, which allows the isolation of nascent DNA chains by affinity chromatography on thiol agarose. The average size of the isolated nascent DNA was 1,000 bp, and more than 80% of the nascent DNA chains had RNA primers at their 5' end. The study of the temporal order of chromosome replication near the origin using this experimental system showed that a segment containing recF and gyrB replicated earlier than a segment containing gyrA and part of the rRNA operon (rrnO). This observation is in agreement with previous in vivo data on the replication of origin region and supports the conclusion that the major activity in our in vitro system was the faithful replication of the ori region.

Bacillus subtilis↗