SEARCH · PubMed Health

Results for “Replicability”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 145 records · Page 8Linked to original sources

PubMed1988

Purification and characterization of replication protein A, a cellular protein required for in vitro replication of simian virus 40 DNA.

The replication of simian virus 40 (SV40) DNA is largely dependent upon cellular replication proteins. To define these proteins we have made use of a cell-free system that is capable of replicating plasmid DNA molecules containing the SV40 origin of replication. Systematic fractionation-reconstitution experiments indicate that there are a minimum of six cellular proteins that are required for efficient viral DNA replication in vitro. We report here the purification of one of these proteins, replication protein A (RP-A), to homogeneity. RP-A is a multisubunit protein that contains four tightly associated polypeptides of 70, 53, 32, and 14 kDa. Partial proteolysis experiments indicate that the 53-kDa polypeptide is closely related to the 70-kDa polypeptide, suggesting that it may be a proteolytic fragment of the larger subunit. RP-A is absolutely required for reconstitution of SV40 DNA replication in vitro. The purified protein binds to single-stranded DNA and is required for the large tumor (T)-antigen-mediated unwinding of DNA molecules containing the SV40 origin of DNA replication. These properties are consistent with the possibility that RP-A plays a central role in the generation of a single-stranded region at the origin prior to initiation of DNA synthesis. The protein may also function to facilitate unwinding of the parental DNA strands during the elongation phase of SV40 DNA replication.

Antigens, Viral, Tumor↗

PubMed1997

recF and recR are required for the resumption of replication at DNA replication forks in Escherichia coli.

Escherichia coli containing a mutation in recF are hypersensitive to UV. However, they exhibit normal levels of conjugational or transductional recombination unless the major pathway (recBC) is defective. This implies that the UV sensitivity of recF mutants is not due to a defect in recombination such as occurs during conjugation or transduction. Here, we show that when replication is disrupted, at least two genes in the recF pathway, recF and recR, are required for the resumption of replication at DNA replication forks, and that in their absence, localized degradation occurs at the replication forks. Our observations support a model in which recF and recR are required to reassemble a replication holoenzyme at the site of a DNA replication fork. These results, when taken together with previous literature, suggest that the UV hypersensitivity of recF cells is due to an inability to resume replication at disrupted replication forks rather than to a defect in recombination. Current biochemical and genetic data on the conditions under which recF-mediated recombination occurs suggest that the recombinational intermediate also may mimic the structure of a disrupted replication fork.

Bacterial Proteins↗

PubMed1996

A replication-enhancing element with transcriptional silencer activity in autonomously replicating human chromosomal fragments.

We have identified specific nucleotide sequences involved in autonomous replication of human chromosomal fragments in human cells. Nested deletion analysis of a 10.2-kb long human chromosomal fragment showed that replication efficiency of the fragment was reduced to about 50% by loss of a short specific segment. Deletions outside the segment reduced the replication efficiency depending on their lengths. By introducing linker substitutions, we found that the distinct segment required for the efficient replication consisted of an 18-bp sequence, named REE1 (Replication Enhancing Element 1). Single or tandem copies of REE1 alone had no significant replication activity, but they stimulated replication of human chromosomal DNA fragments. We found, in addition, that the REE1 sequence inserted at a site 2.7 kb upstream of the SV40 early promoter caused repression of transcription from the promoter, suggesting that REE1 had a transcriptional silencer activity. Introduction of linker substitutions into the REE1 indicated that the nucleotide sequences required for the repression of transcription were the same as those for enhancement of replication. Thus, REE1 is responsible for both enhancement of replication and repression of transcription.

Base Sequence↗

PubMed1994

Evolution of the replication regions of IncI alpha and IncFII plasmids by exchanging their replication control systems.

The basic replicons of bacterial plasmids consist of two sets of genetic systems, the replication-structural system and the replication control system. Comparison of nucleotide sequences suggested that the basic replicons of plasmids P307 (IncFI) and pMU2200 (IncZ) were generated by reciprocal recombination between ancestors of R100 (IncFII) and ColIb-P9 (IncI alpha), or vice versa. The plasmids of each pair, P307/pMU2200 and R100/ColIb-P9, are structurally unrelated to each other. Based on this information, we constructed in vitro and analyzed P307-like chimeric replicons from ColIb-P9 and R100. When the replication-structural region of ColIb-P9 was combined with the whole replication control region of R100, the resultant replicon replicated stably as R100 did. These results revealed that the basic replicons of the plasmids diverged by exchanging their replication control systems. Thus, we propose that the replication control systems of plasmids, in some cases, evolved independently of their structural systems, although these two systems work together to maintain the replication functions. We also showed that the reciprocal recombination was specified by the unique secondary structures of RNA involved in the control of expression of the genes encoding the replication initiator proteins.

Amino Acid Sequence↗

PubMed1990

Autonomous replication of a DNA fragment containing the chromosomal replication origin of the human c-myc gene.

The c-myc genes of HeLa cells are preferentially replicated in the transcriptional direction, from chromosomal origin sequences which display cell type-specific activity. Using a run-off replication assay involving in vitro extension of replication forks initiated in intact HeLa cells, bidirectional replication was observed to begin within a 3.5 kb domain 5' to the c-myc gene. To characterize the replication origin further a 2.4 HindIII-Xhol subfragment of the c-myc 5' flanking DNA was cloned in a selectable vector and transfected into HeLa cells. The resulting pNeo.Myc-2.4 construct persisted as a circular extrachromosomal element for more than 300 cell generations under selection, with recovery of approximately 500-1000 times the mass of plasmid initially introduced into the cells. Extrachromosomal circular pNeo.Myc-2.4 monomer was reisolated in supercoiled form, along with oligomeric and miniplasmid variants which had been generated in vivo; however, chromosomally integrated copies of the plasmid were not detectable in cultures containing extrachromosomal pNeo.Myc-2.4. The recovered pNeo.Myc-2.4 plasmid was resistant to Dpnl digestion and sensitive to Mbol digestion. After transfection with pNeo.Myc-2.4 BrUdR pulse labeling of long-term or short-term cultures demonstrated that the plasmid replicated semiconservatively, under controls similar to those imposed on chromosome replication. Bisection of the pNeo.Myc-2.4 insert suggested that c-myc 5' flanking DNA within 1.2 kb 5' to promoter P1 was sufficient to confer autonomously replicating sequence activity on the plasmid vector in transient replication assays.

Chromosomes, Human↗

PubMed2005

Replication checkpoint kinase Cds1 regulates Mus81 to preserve genome integrity during replication stress.

The replication checkpoint kinase Cds1 preserves genome integrity by stabilizing stalled replication forks. Cds1 targets substrates through its FHA domain. The Cds1 FHA domain interacts with Mus81, a subunit of the Mus81-Eme1 structure-specific endonuclease. We report here that Mus81 and Rhp51 are required for generating deletion mutations in fission yeast replication mutants that experience replication stress. A mutation in the Mus81 FHA-binding motif eliminates its Cds1-binding and Cds1-dependent phosphorylation. Furthermore, this mutation exacerbates the deletion mutator phenotype of a replication mutant, and induces a hyper-recombination phenotype in hydroxyurea-treated cells. In unperturbed cells, Mus81 associates with chromatin throughout S phase. In replication mutants grown at semipermissive temperature, Mus81 undergoes minor Cds1-dependent phosphorylation, remains chromatin-associated, generates deletion mutations, and maintains cell growth. Upon S-phase arrest by acute hydroxyurea treatment, Mus81 is not required for cell viability but is essential for recovery from replication fork collapse. Moreover, Mus81 undergoes extensive Cds1-dependent phosphorylation and dissociates from chromatin in hydroxyurea-arrested cells, thereby preventing it from cleaving stalled replication forks that could lead to fork breakage and chromosomal rearrangement. These results provide novel insights into how Cds1 regulates Mus81 accordingly when cells experience different replication stress to preserve genome integrity.

Checkpoint Kinase 2↗

PubMed2001

A DNA replication-arrest site RTS1 regulates imprinting by determining the direction of replication at mat1 in S. pombe.

Mating-type switching in Schizosaccharomyces pombe involves a strand-specific, alkali-labile imprint at the mat1 (mating-type) locus. The imprint is synthesized during replication in a swi1, swi3, and polymerase alpha (swi7) dependent manner and is dependent on mat1 being replicated in a specific direction. Here we show that the direction of replication at mat1 is controlled by a cis-acting polar terminator of replication (RTS1). Two-dimensional gel analysis of replication intermediates reveals that RTS1 only terminates replication forks moving in the centromere-distal direction. A genetic analysis shows that RTS1 optimizes the imprinting process. Transposing the RTS1 element to the distal side of mat1 abolishes imprinting of the native mat1 allele but restores imprinting of an otherwise unimprinted inverted mat1 allele. These data provide conclusive evidence for the "direction of replication model" that explains the asymmetrical switching pattern of S. pombe, and identify a DNA replication-arrest element implicated in a developmental process. Such elements could play a more general role during development and differentiation in higher eukaryotes by regulating the direction of DNA replication at key loci.

Base Sequence↗

PubMed2006

Proteolysis of the replication checkpoint protein Sda is necessary for the efficient initiation of sporulation after transient replication stress in Bacillus subtilis.

Cells of Bacillus subtilis actively co-ordinate the initiation of sporulation with DNA replication and repair. Conditions that perturb replication initiation or replication elongation induce expression of a small protein, Sda, that specifically inhibits the histidine kinases required to initiate spore development. Previously, the role of Sda has been studied during chronic blocks to DNA replication. Here we show that induction of Sda is required to delay the initiation of sporulation when replication elongation is transiently blocked or after UV irradiation. During the recovery phase, cells efficiently sporulated, but this required the proteolysis of Sda. The rapid proteolysis of Sda required the ClpXP protease and the uncharged C-terminal sequence of Sda. Replacing the last two residues of Sda, both serines, with aspartic acids markedly stabilized Sda. Strains expressing sdaDD from the endogenous sda locus were unable to efficiently initiate sporulation after transient replication stress. We conclude that the Sda replication checkpoint is required to delay the initiation of sporulation when DNA replication is transiently perturbed, and that the intrinsic instability of Sda contributes to shutting off the pathway. The Sda checkpoint thus co-ordinates early events of spore development, including the polar cell division, with successful completion of chromosome replication.

Amino Acid Sequence↗

PubMed2006

Replication of hepatitis C virus (HCV) RNA in mouse embryonic fibroblasts: protein kinase R (PKR)-dependent and PKR-independent mechanisms for controlling HCV RNA replication and mediating interferon activities.

Hepatitis C virus (HCV) infection causes chronic hepatitis and is currently treated with alpha interferon (IFN-alpha)-based therapies. The underlying mechanisms of chronic HCV infection and IFN-based therapies, however, have not been defined. Protein kinase R (PKR) was implicated in the control of HCV replication and mediation of IFN-induced antiviral response. In this report, we demonstrate that a subgenomic RNA replicon of genotype 2a HCV replicated efficiently in mouse embryonic fibroblasts (MEFs), as determined by cell colony formation efficiency and the detection of HCV proteins and both positive- and negative-strand RNAs. Additionally, the subgenomic HCV RNA was found to replicate more efficiently in the PKR knockout (PKR(-/-)) MEF than in the wild-type (PKR(+/+)) MEF. The knockdown expression of PKR by specific small interfering RNAs significantly enhanced the level of HCV RNA replication, suggesting that PKR is involved in the control of HCV RNA replication. The level of ISG56 (p56) was induced by HCV RNA replication, indicating the activation of PKR-independent antiviral pathways. Furthermore, IFN-alpha/beta inhibited HCV RNA replication in PKR(-/-) MEFs as efficiently as in PKR(+/+) MEFs. These findings demonstrate that PKR-independent antiviral pathways play important roles in controlling HCV replication and mediating IFN-induced antiviral effect. Our findings also provide a foundation for the development of transgenic mouse models of HCV replication and set a stage to further define the roles of cellular genes in the establishment of chronic HCV infection and the mediation of intracellular innate antiviral response by using MEFs derived from diverse gene knockout animals.

Animals↗

PubMed2007

Mutations that decrease DNA binding of the processivity factor of the herpes simplex virus DNA polymerase reduce viral yield, alter the kinetics of viral DNA replication, and decrease the fidelity of DNA replication.

The processivity subunit of the herpes simplex virus DNA polymerase, UL42, is essential for viral replication and possesses both Pol- and DNA-binding activities. Previous studies demonstrated that the substitution of alanine for each of four arginine residues, which reside on the positively charged surface of UL42, resulted in decreased DNA binding affinity and a decreased ability to synthesize long-chain DNA by the polymerase. In this study, the effects of each substitution on the production of viral progeny, viral DNA replication, and DNA replication fidelity were examined. Each substitution mutant was able to complement the replication of a UL42 null mutant in transient complementation assays and to support the replication of plasmid DNA containing herpes simplex virus type 1 (HSV-1) origin sequences in transient DNA replication assays. Mutant viruses containing each substitution and a lacZ insertion in a nonessential region of the genome were constructed and characterized. In single-cycle growth assays, the mutants produced significantly less progeny virus than the control virus containing wild-type UL42. Real-time PCR assays revealed that these UL42 mutants synthesized less viral DNA during the early phase of infection. Interestingly, during the late phase of infection, the mutant viruses synthesized larger amounts of viral DNA than the control virus. The frequencies of mutations of the virus-borne lacZ gene increased significantly in the substitution mutants compared to those observed for the control virus. These results demonstrate that the reduced DNA binding of UL42 is associated with significant effects on virus yields, viral DNA replication, and replication fidelity. Thus, a processivity factor can influence replication fidelity in mammalian cells.

Animals↗

PubMed1979

Late replicative intermediates are accumulated during simian virus 40 DNA replication in vivo and in vitro.

Simian virus 40 (SV40) replicating chromosomes were extracted from nuclei of infected cells. The chromosomes in the extract were resolved on neutral sucrose gradients, and the extent of replication of the DNA in the chromosome peaks was determined. The extract, in combination with cytosol factors and the appropriate precursors, supports the continued replication of viral DNA. The products of the incubation were mature form I DNA and molecules (after deproteinization) with sedimentation coefficients, in neutral sucrose, of 22S and 29S. The results of our analysis of this system indicate the following. (i) The 22S molecule, which has been described by previous workers, is a relaxed, replicating molecule and is an artifact of the in vitro system. (ii) When the in vitro synthesis is performed at optimal ionic strength (150 mM potassium acetate), the artifactual 22S molecule does not appear. (iii) Late replicative intermediates do accumulate in vivo and in vitro. The major late form accumulated is 91% completed. (iv) The replicating chromosomes can be resolved into two distinct peaks on neutral sucrose gradients. The molecules in these peaks differ in extent of replication. (v) The nuclear extraction procedure preferentially extracts early replicating chromosomes. The relevance of these data to the problem of SV40 and cellular chromosome replication and termination is described.

Centrifugation, Density Gradient↗

PubMed2003

New reporter gene-based replication assay reveals exchangeability of replication factors of porcine circovirus types 1 and 2.

Two types of porcine circovirus (PCV), which differ in their pathogenicity, are known. PCV type 2 (PCV2) is the etiological agent of postweaning multisystemic wasting syndrome in swine, while PCV1 has not yet been linked to a disease. Corroborating earlier observations in PCV1, transcript mapping revealed that the rep gene of PCV2 encodes two products, the full-length protein Rep and the spliced version Rep' and that the simultaneous expression of Rep and Rep' proteins is essential for initiation of replication of PCV2. The interchangeability of the replication factors of PCV1 and PCV2 was examined. The rep gene products of PCV2 were not only able to bind the PCV2 origin but also the origin of PCV1 and vice versa. To investigate the competence of the Rep/Rep' proteins to initiate replication at the heterologous origin, a new replication assay was developed. It measures the expression of a luc reporter gene present on a plasmid carrying the origin of the investigated replicon. Replication is initiated by expression of the appendant replicase from a second plasmid and results in replication of the origin plasmid coupled with an increase in the Luc activity. Using this method to compare replication of PCV1 and PCV2 in cell culture, it was shown that the Rep/Rep' protein of PCV2 initiated replication at the origin of PCV1, as did the reciprocal combination. Our results indicate that the cis- and trans-acting replication factors of the two viruses are functionally exchangeable.

Animals↗

PubMed2004

Assembly of turnip yellow mosaic virus replication complexes: interaction between the proteinase and polymerase domains of the replication proteins.

Turnip yellow mosaic virus (TYMV), a positive-strand RNA virus in the alphavirus-like supergroup, encodes two nonstructural replication proteins (140K and 66K), both of which are required for its RNA genome replication. The 140K protein contains domains indicative of methyltransferase, proteinase, and NTPase/helicase activities, while the 66K protein encompasses the RNA-dependent RNA polymerase domain. Recruitment of the 66K protein to the sites of viral replication, located at the periphery of chloroplasts, is dependent upon the expression of the 140K protein. Using antibodies raised against the 140K and 66K proteins and confocal microscopy, we report the colocalization of the TYMV replication proteins at the periphery of chloroplasts in transfected or infected cells. The replication proteins cofractionated in functional replication complexes or with purified chloroplast envelope membranes prepared from infected plants. Using a two-hybrid system and coimmunoprecipitation experiments, we also provide evidence for a physical interaction of the TYMV replication proteins. In contrast to what has been found for other members of the alphavirus-like supergroup, the interaction domains were mapped to the proteinase domain of the 140K protein and to a large region encompassing the core polymerase domain within the 66K protein. Coexpression and colocalization experiments confirmed that the helicase domain of the 140K protein is unnecessary for the proper recruitment of the 66K protein to the chloroplast envelope, while the proteinase domain appears to be essential for that process. These results support a novel model for the interaction of TYMV replication proteins and suggest that viruses in the alphavirus-like supergroup may have selected different pathways to assemble their replication complexes.

Arabidopsis↗

PubMed1991

Replication initiates at multiple locations on an autonomously replicating plasmid in human cells.

We have used a two-dimensional gel electrophoresis mapping technique to determine where DNA replication initiates on a plasmid which utilizes a fragment of human DNA to replicate autonomously in human cells. Replication was found to initiate at multiple locations on the plasmid carrying the human sequence, in contrast to the pattern seen for an Epstein-Barr virus vector which served as a control with a fixed origin. The family of repeats, a portion of the Epstein-Barr virus origin of replication which is present our plasmid, was shown to function as a replication fork barrier. The nature of the stalled replicative intermediates on the human DNA-based plasmid further indicated that replication did not initiate at a single fixed position each time the plasmid replicated. The results suggest that the replication apparatus used to duplicate DNA in human cells may not have precise sequence requirements which target initiation to specific locations.

Cell Cycle↗

PubMed1992

Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand DNA replication complex.

A number of proteins have been isolated from human cells on the basis of their ability to support DNA replication in vitro of the simian virus 40 (SV40) origin of DNA replication. One such protein, replication factor C (RFC), functions with the proliferating cell nuclear antigen (PCNA), replication protein A (RPA), and DNA polymerase delta to synthesize the leading strand at a replication fork. To determine whether these proteins perform similar roles during replication of DNA from origins in cellular chromosomes, we have begun to characterize functionally homologous proteins from the yeast Saccharomyces cerevisiae. RFC from S. cerevisiae was purified by its ability to stimulate yeast DNA polymerase delta on a primed single-stranded DNA template in the presence of yeast PCNA and RPA. Like its human-cell counterpart, RFC from S. cerevisiae (scRFC) has an associated DNA-activated ATPase activity as well as a primer-template, structure-specific DNA binding activity. By analogy with the phage T4 and SV40 DNA replication in vitro systems, the yeast RFC, PCNA, RPA, and DNA polymerase delta activities function together as a leading-strand DNA replication complex. Now that RFC from S. cerevisiae has been purified, all seven cellular factors previously shown to be required for SV40 DNA replication in vitro have been identified in S. cerevisiae.

Adenosine Triphosphatases↗

PubMed1994

Transcription inhibits the replication of autonomously replicating plasmids in human cells.

This study addresses the effect of transcription on replication, using a system based on autonomously replicating plasmids in human cells. We added transcriptional elements from the human cytomegalovirus promoter/enhancer and the human beta-actin promoter to autonomously replicating plasmids based on human sequences and found that the transcriptional elements inhibited plasmid replication. Furthermore, conditional inhibition of plasmid replication was demonstrated by using a tetracycline-responsive promoter. We found that replication activity of plasmids carrying this promoter was inversely correlated with promoter activity. Replication activity was partially restored on plasmids when a transcriptional termination sequence was placed directly downstream of the promoter element. Transcriptional activity of the promoters and the efficacy of the terminator sequence were confirmed by using steady-state RNA analysis. These experiments suggest that transcription inhibits DNA replication on these plasmids and that the degree of inhibition is dependent on transcription strength. The possible significance of these results for chromosomal DNA replication are discussed.

Actins↗

PubMed1996

Induction of DNA replication by transcription in the region upstream of the human c-myc gene in a model replication system.

An important relationship between transcription and initiation of DNA replication in both eukaryotes and prokaryotes has been suggested. In an attempt to understand the molecular mechanism of this interaction, we examined whether transcription can induce DNA replication in vitro by constructing a system in which both replication and transcription were combined. Relaxed circular DNA possessing a replication initiation zone located upstream of the human c-myc gene and a T7 promoter near the P1 promoter of the gene was replicated in the presence of T7 RNA polymerase. In our model system, replication was carried out with the proteins required for simian virus 40 DNA replication. DNA synthesis, which was dependent on both T7 RNA polymerase and the replication proteins, was detected mainly in the promoter and upstream regions of the c-myc gene. Blocking RNA synthesis at the initial stage of the reaction severely reduced DNA synthesis, suggesting that RNA chain elongation is required to induce DNA synthesis. The results indicated that transcription can induce DNA replication in the upstream region of the transcribed gene, most likely by introducing negative supercoiling into the region, which results in unwinding of the DNA duplex.

Bacteriophage T7↗

PubMed2006

Combination therapy of androgen-independent prostate cancer using a prostate restricted replicative adenovirus and a replication-defective adenovirus encoding human endostatin-angiostatin fusion gene.

Although prostate-restricted replicative adenovirus has exhibited significant antitumor efficacy in preclinical studies, it is necessary to develop more potent adenoviruses for prostate cancer gene therapy. We evaluated the synergistic killing effect of prostate-restricted replicative adenovirus and AdEndoAngio, a replication-defective adenovirus expressing the endostatin-angiostatin fusion protein (EndoAngio). When coadministered with AdEndoAngio, prostate-restricted replicative adenovirus significantly elevated EndoAngio expression, suggesting that AdEndoAngio coreplicates with prostate-restricted replicative adenovirus. Conditioned medium from prostate cancer cells infected by prostate-restricted replicative adenovirus plus AdEndoAngio inhibited the growth, tubular network formation, and migration of human umbilical vein endothelial cells better than conditioned medium from prostate cancer cells infected by AdEndoAngio alone. Furthermore, in vivo animal studies showed that the coadministration of prostate-restricted replicative adenovirus plus AdEndoAngio resulted in the complete regression of seven out of eight treated androgen-independent CWR22rv tumors, with a tumor nodule maintaining a small size for 14 weeks. The residual single tumor exhibited extreme pathologic features together with more endostatin-reactive antibody-labeled tumor cells and fewer CD31-reactive antibody-labeled capillaries than the AdEndoAngio-treated tumors. These results show that combination therapy using prostate-restricted replicative adenovirus together with antiangiogenic therapy has more potent antitumor effects and advantages than single prostate-restricted replicative adenovirus and deserves more extensive investigation.

Adenoviridae↗