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At least 253 records · Page 14Linked to original sources

PubMed1994

Reconstitution of complete SV40 DNA replication with purified replication factors.

The identification and purification of human cell proteins required for the production of form I DNA following DNA replication from the simian virus 40 (SV40) origin is described. Using these proteins, complete SV40 DNA replication was reconstituted with only purified DNA replication factors: SV40 large tumor antigen (TAg), replication protein A (RPA), DNA topoisomerases I and II, DNA polymerase alpha-primase, replication factor C (RFC), the proliferating cell nuclear antigen (PCNA), DNA polymerase delta, maturation factor 1 (MF1), and DNA ligase I. MF1, a 5' to 3' exonuclease and DNA ligase I were both identified as essential components for production of covalently closed circular relaxed (form I) DNA. MF1 is probably the same exonuclease previously shown by others to function during DNA synthesis on artificial DNA templates or in conjunction with DNA polymerase alpha from the SV40 origin. Combined with these previous studies, our results suggest that MF1 functions to remove an RNA primer attached to every Okazaki fragment during lagging strand DNA synthesis. Interestingly, whereas mammalian DNA ligase I functioned in the reconstituted replication system, mammalian DNA ligase III did not substitute and the phage T4 DNA ligase functioned inefficiently, suggesting that DNA ligase I has a specific role as a replicative DNA ligase in eukaryotic cells.

Animals↗

PubMed2005

Replication-coupled modulation of early replicating chromatin domains detected by anti-actin antibody.

Evidence is presented for the reversible, cold-dependent immunofluorescence detection of the epitope (hereafter referred to as epiC), recognized by a monoclonal anti-actin antibody in diploid human fibroblast cell nuclei and mitotic chromosomes. The nuclear/chromosomal epiC was detected in a cell cycle window beginning in early S phase and extending through S phase, G(2) phase, mitosis until early G(1) phase of the subsequent daughter cells. A small but significant level of co-localization was measured between the nuclear epiC and active sites of DNA replication in early S phase. The level of co-localization was strikingly enhanced beginning approximately 1 h after the initial labeling of early S phase replicating chromatin domains. In contrast, epiC did not co-localize with late S phase replicated chromatin either during DNA replication or at any other time in the cell cycle. We propose a replication-coupled modulation of early S phase replicated chromatin domains that is detected by the chromatin epiC positivity, persists on the chromatin domains from early S until early G(1) of the next cell generation, and may be involved in the regulation and/or coordination of replicational and transcriptional processes during the cell cycle. Further studies will be required to resolve the possible role of nuclear actin in this modulation process.

Actins↗

PubMed2007

Chlorpromazine and apigenin reduce adenovirus replication and decrease replication associated toxicity.

BACKGROUND: Adenoviruses can cause severe toxicity in immunocompromised individuals. Although clinical trials have confirmed the potency and safety of selectively oncolytic adenoviruses for treatment of advanced cancers, increasingly effective agents could result in more toxicity and therefore it would be useful if replication could be abrogated if necessary. METHODS: We analyzed the effect of chlorpromazine, an inhibitor of clathrin-dependent endocytosis and apigenin, a cell cycle regulator, on adenovirus replication and toxicity. First, we evaluated the in vitro replication of a tumor targeted Rb-p16 pathway selective oncolytic adenovirus (Ad5/3-Delta24) and a wild-type adenovirus in normal cells, fresh liver samples and in ovarian cancer cell lines. Further, we analyzed the in vitro cell killing efficacy of adenoviruses in the presence and absence of the substances. Moreover, the effect on in vivo efficacy, replication and liver toxicity of the adenoviruses was evaluated. RESULTS: We demonstrate in vitro and in vivo reduction of adenovirus replication and associated toxicity with chlorpromazine and apigenin. Effective doses were well within what would be predicted safe in humans. CONCLUSIONS: Chlorpromazine and apigenin might reduce the replication of adenovirus, which could provide a safety switch in case replication-associated side effects are encountered in patients. In addition, these substances could be useful for the treatment of systemic adenoviral infections in immunosuppressed patients.

Adenocarcinoma↗

PubMed1995

Identification of essential trans-acting regions required for DNA replication of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus: lef-1 is an essential replication gene.

A transient replication assay for the identification of baculovirus genes that are essential for replication of an origin-containing reporter plasmid was established for the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV). Using a replication origin located on the OpMNPV HindIII-N fragment, we identified a subset of cosmids and plasmids from an OpMNPV cosmid library that was able to supply all the essential trans-acting factors and support replication of the origin-containing plasmid in uninfected Lymantria dispar cells. However, this limited set of DNA's was unable to support replication of a second origin-containing plasmid derived from a different region of the OpMNPV genome. Replication analysis of deletion clones of the HindIII-N fragment led to the identification of a gene, late expression factor 1 (lef-1), that is essential for the transactivation of DNA replication in this system. Transcriptional analysis of lef-1 mapped both early and late transcripts of about 1.75 kb. A motif characteristic of nucleoside triphosphate-binding sites present in the carboxy-terminal region of AcMNPV Lef-1 is not conserved in OpMNPV Lef-1.

Amino Acid Sequence↗

PubMed2002

Replication of Potato spindle tuber viroid in cultured cells of tobacco and Nicotiana benthamiana: the role of specific nucleotides in determining replication levels for host adaptation.

We have developed an electroporation protocol to inoculate cultured cells of tobacco and Nicotiana benthamiana with in vitro transcripts of Potato spindle tuber viroid (PSTVd) to characterize viroid structural features that determine replication efficiency at the cellular level. Both (+)- and (-)-strands of PSTVd were detected by Northern blots as early as 6 h postinoculation (h.p.i.). Accumulation of the (+)-circular PSTVd increased very rapidly starting at 24 h.p.i. and continued beyond 6 days postinoculation. Viroid accumulation in individual cells was visualized by in situ hybridization, which showed that 60-70% of the cells were infected. Previous work showed that C259 --> U substitution converted tomato isolate PSTVd(KF440-2) into a strain that is infectious on tobacco (M. Wassenegger, R. L. Spieker, S. Thalmeir, F.-U. Gast, L. Riedel, and H. L. Sänger, 1996. Virology 226, 191-197). Similarly, C259 --> U or U257 --> A substitution in the Intermediate strain (PSTVd(Int)) conferred infectivity in tobacco (Y. Zhu, Y. Qi, Y. Xun, R. Owens, and B. Ding, 2002. Plant Physiol. 130, 138-146). Our replication assays in tobacco-cultured cells demonstrated that U257 --> A and C259 --> U substitutions each enhanced PSTVd replication by 5- to 10-fold. Replacement of U257 with C, but not with G, also led to enhanced replication in tobacco cells. Replacement of C259 with nucleotide A or G did not enhance replication. Elevated accumulation of the (-)- and (+)-strands of these mutants was in part due to enhanced transcription. Interestingly, all of the nucleotide changes did not alter PSTVd replication levels in N. benthamiana cells. These results provide insights about PSTVd structures that modulate replication efficiency in adapting to a specific host.

Base Sequence↗

PubMed1987

5-aza-C-induced changes in the time of replication of the X chromosomes of Microtus agrestis are followed by non-random reversion to a late pattern of replication.

Treatment with 5-azacytidine (5-aza-C) causes an advance in the time of replication and enhances the DNase-I sensitivity of the inactive X chromosome in Gerbillus gerbilllus fibroblasts. We found that these changes were not stably inherited and upon removal of the drug the cells reverted to the original state of one active and one inactive X chromosome. In order to determine whether this reversion was random, we used a cell line of female Microtus agrestis fibroblasts in which the two X chromosomes are morphologically distinguishable. In this work we show that the reversion to a late pattern of replication is not random, and the originally late replicating X chromosome is preferentially "reinactivated", suggesting an imprinting-like marking of one or both X chromosomes. The changes in the replication pattern of the X chromosome were associated with changes in total DNA methylation. Double treatment of cells with 5-aza-C did not alter this pattern of euchromatin activation and reinactivation. A dramatic advance in the time of replication of the entire X linked constitutive heterochromatin (XCH) region was however, observed in the doubly treated cells. This change in the replication timing of the XCH occurred in both X chromosomes and was independent of the changes observed in the euchromatic region. These observations suggest the existence of at least two independent regulatory sites which control the timing of replication of two large chromosomal regions.

Animals↗

PubMed1988

Replication of ColE2 and ColE3 plasmids: the regions sufficient for autonomous replication.

We have localized the regions sufficient for autonomous replication on the genomes of the colicin E2 (ColE2) and colicin E3 (ColE3) plasmids and analyzed the replication functions carried by these regions. A 1.3 kb segment of each plasmid is sufficient for autonomous replication. Plasmids carrying this segment retain the replication properties of the original plasmid. The 1.3 kb segment consists of three functional portions. Firstly, a 0.9 kb region which specifies at least one trans-acting factor required for replication of each plasmid. Secondly, a 0.4 kb region located adjacent to one end of the 0.9 kb region, which is required for expression of the trans-acting factor(s) and probably contains the promoter. The region across the border of these two portions of ColE2 is involved in copy number control of the plasmid. The third portion is a 50 bp region adjacent to the other end of the 0.9 kb region, which contains a cis-acting site (origin) where replication initiates in the presence of the trans-acting factor(s). The action of the trans-acting factor(s) on the origin is plasmid specific. The 50 bp regions functioning as the origins of replication of ColE2 and ColE3 are the smallest among those in prokaryotic replicons so far identified and analyzed.

Colicins↗

PubMed1987

E. coli minichromosome replication in vitro and in vivo: comparative analyses of replication intermediates.

The process of replication of Escherichia coli minichromosomes was examined by following the intermediates formed in vitro and in vivo. Replication initiated on a supercoiled closed circular (CC) monomer, proceeded rapidly to a late but incomplete stage in polymerization (the LC form) in both systems, passed more slowly through a series of open and closed circular catenated dimers with varying extents of intertwining between the monomer units, and then yielded, after decatenation, the supercoiled CC monomer. The replication patterns of two different minichromosomes were similar, although the LC form and the multiply intertwined dimers were much more evident in the smaller pAL4 than in pAL2. The same basic replication scheme was seen in vitro and in vivo but completion of polymerization and processing of the dimers were slower in vitro. Some radioactivity was detected in OC monomer early during replication, consistent with occasional decatenation of LC structures to produce OC molecules which then completed replication to form CC molecules. However, progression to CC catenated dimers prior to formation of CC monomers represented the major replication pathway.

Centrifugation, Density Gradient↗

PubMed1993

Identification of the initiation region of DNA replication in the murine immunoglobulin heavy chain gene and possible function of the octamer motif as a putative DNA replication origin in mammalian cells.

An origin region of DNA replication in the murine immunoglobulin heavy chain (IgH) gene was identified by BrdU pulse labeling and PCR amplification methods. The origin region spans about 1000 base pairs and contains the region of transcriptional enhancer in which the octamer sequence is present. The octamer sequence, TNATTTGCAT, is a well-conserved promoter/enhancer element responsible for B cell-specific transcription and is also found in the regulatory sequences for histone genes and others. Its activity as an autonomously replicating sequence was further examined. The murine IgH enhancer region containing the octamer motif was cloned in pUC18 and transfected to HeLa cells. After 60-65 h, the low molecular weight DNA was extracted and the degree to which the plasmid DNA had been replicated in the cells was measured by back-transformation of competent bacteria. Five to ten copies of the plasmid were detected per cell. The replicated plasmid-form DNA could be detected by this assay for at least 7 days after transfection. Synthetic oligonucleotides corresponding to the octamer and the Ephrussi box in the IgH enhancer were also cloned into pUC18 and examined for replicating activity. These plasmids replicated provided that the octamer sequence remained intact, irrespective of the Ephrussi box sequence and of the sites of insertion. These results suggest that the octamer transcriptional element may also serve as a putative origin for cellular DNA replication.

Animals↗

PubMedDate not supplied

Xenopus ATR is a replication-dependent chromatin-binding protein required for the DNA replication checkpoint.

BACKGROUND: The DNA replication checkpoint ensures that mitosis is not initiated before DNA synthesis is completed. Recent studies using Xenopus extracts have demonstrated that activation of the replication checkpoint and phosphorylation of the Chk1 kinase are dependent on RNA primer synthesis by DNA polymerase alpha, and it has been suggested that the ATR kinase-so-called because it is related to the product of the gene that is mutated in ataxia telangiectasia (ATM) and to Rad3 kinase-may be an upstream component of this response. It has been difficult to test this hypothesis as an ATR-deficient system suitable for biochemical studies has not been available. RESULTS: We have cloned the Xenopus laevis homolog of ATR (XATR) and studied the function of the protein in Xenopus egg extracts. Using a chromatin-binding assay, we found that ATR associates with chromatin after initiation of replication, dissociates from chromatin upon completion of replication, and accumulates in the presence of aphidicolin, an inhibitor of DNA replication. Its association with chromatin was inhibited by treatment with actinomycin D, an inhibitor of RNA primase. There was an early rise in the activity of Cdc2-cyclin B in egg extracts depleted of ATR both in the presence or absence of aphidicolin. In addition, the premature mitosis observed upon depletion of ATR was accompanied by the loss of Chk1 phosphorylation. CONCLUSIONS: ATR is a replication-dependent chromatin-binding protein, and its association with chromatin is dependent on RNA synthesis by DNA polymerase alpha. Depletion of ATR leads to premature mitosis in the presence and absence of aphidicolin, indicating that ATR is required for the DNA replication checkpoint.

Amino Acid Sequence↗

PubMed1999

Evidence that a single replication fork proceeds from early to late replicating domains in the IgH locus in a non-B cell line.

In non-B cell lines, like the murine erythroleukemia cell line (MEL), the most distal IgH constant region gene, C alpha, replicates early in S; other heavy chain constant region genes, joining and diversity segments, and the most proximal Vh gene replicate successively later in S in a 3' to 5' direction proportional to their distance from C alpha. In MEL, replication forks detected in the IgH locus also proceed in the same 3' to 5' direction for approximately 400 kb, beginning downstream of the IgH 3' regulatory region and continuing to the D region, as well as within the Vh81X gene. Downstream of the initiation region is an early replicating domain, and upstream of Vh81X is a late replicating domain. Hence, the gradual transition between early and late replicated domains can be achieved by a single replication fork.

Animals↗

PubMed2000

Interactions between Mcm10p and other replication factors are required for proper initiation and elongation of chromosomal DNA replication in Saccharomyces cerevisiae.

BACKGROUND: MCM10 is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae. Previous work showed that Mcm10p interacts with the Mcm2-7 protein complex that may be functioning as the replication-licensing factor. In addition, Mcm10p is required during origin activation and disassembly of the prereplicative complex, which allows smooth passage of replication forks. RESULTS: We show that an mcm10 mutation causes a slow progression of DNA synthesis and a loss of chromosome integrity during the S phase and prevents entry into mitosis, despite apparent completion of chromosomal DNA replication at nonpermissive temperatures. Furthermore, Mcm10p interacts genetically with the origin recognition complex (ORC) and various replication elongation factors, including a subunit of DNA polymerases epsilon and delta. Mcm10p is an abundant protein (approximately 4 x 10(4) copies per haploid cell) that is almost exclusively localized in the chromatin and/or nuclear matrix fractions during all phases of the cell cycle. When it is visualized by the chromosome-spreading method followed by immunostaining, Mcm10p forms punctate foci on chromatin throughout the cell cycle and these foci mostly overlap with those of Orc1p, a component of ORC. CONCLUSIONS: These results suggest that Mcm10p, like the Mcm2-7 proteins, is a critical component of the prereplication chromatin and acts together with ORC during the initiation of chromosomal DNA replication; in addition, Mcm10p plays an important role during the elongation of DNA replication.

Cell Cycle Proteins↗

PubMed1999

The replication checkpoint control in Bacillus subtilis: identification of a novel RTP-binding sequence essential for the replication fork arrest after induction of the stringent response.

We have shown previously that induction of the stringent response in Bacillus subtilis resulted in the arrest of chromosomal replication between 100 and 200 kb either side of oriC at distinct stop sites, designated LSTer and RSTer, left and right stringent terminators respectively. This replication checkpoint was also shown to involve the RTP protein, normally active at the chromosomal terminus. In this study, we show that the replication block is absolutely dependent upon RelA, correlated with high levels of ppGpp, but that efficient arrest at STer sites also requires RTP. DNA-DNA hybridization data indicated that one or more such LSTer sites mapped to gene yxcC (-128 kb from oriC). A 7.75 kb fragment containing this gene was cloned into a theta replicating plasmid, and plasmid replication arrest, requiring both RelA and RTP, was demonstrated. This effect was polar, with plasmid arrest only detected when the fragment was orientated in the same direction with respect to replication, as in the chromosome. This LSTer2 site was further mapped to a 3.65 kb fragment overlapping the next40 probe. Remarkably, this fragment contains a 17 bp sequence (B'-1) showing 76% identity with an RTP binding site (B sequence) present at the chromosomal terminus. This B'-1 sequence, located in the gene yxcC, efficiently binds RTP in vitro, as shown by DNA gel retardation studies and DNase I footprinting. Importantly, precise deletion of this sequence abolished the replication arrest. We propose that this modified B site is an essential constituent of the LSTer2 site. The differences between arrest at the normal chromosomal terminus and arrest at LSTer site are discussed.

Bacillus subtilis↗

PubMed1972

Relationship of Flac replication and chromosome replication.

The time of replication of a bacterial plasmid, Flac, during the division cycle of Escherichia coli has been estimated in exponentially growing cultures and at various times after a shift from minimal medium to a richer medium (a shift-up). There is a variation in the cell age at which the capacity to synthesize beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) doubles (assumed to be a measure of the time at which the Flac plasmid replicates) when this capacity is measured at various times during the shift-up, and with increasing steady-state exponential growth rate. Cells growing at slow and moderate growth rates exhibit Flac replication in the middle of the division cycle. With increasing time after a shift-up or with increasing growth rate the plasmid replicates at earlier times, eventually at cell division, and finally in the older cells. This variation in the cell age at which the plasmid replicates is similar to the variation in cell age at which chromosome initiation occurs during a shift-up, although plasmid replication occurs slightly before initiation of chromosome replication.

Amino Acids↗

PubMed1974

Replication of colicin E1 plasmid DNA in minicells from a unique replication initiation site.

Replicating DNA molecules of the colicin E1 plasmid isolated from minicells are cleaved at a single site by R1 restriction endonuclease (EcoR1). Electron microscopic measurements of the replicating molecules treated with the endonuclease indicate that (a) replication is initiated at a site between 14% and 20% of the distance from the EcoR1 endonuclease cleavage site; and (b) extensive replication of most molecules occurs in one direction from the initiation site, although a limited amount of replication in the opposite direction may occur. Single-stranded regions at one or both replication forks, involving one or both DNA strands, can be frequently found in replicating molecules.

Colicins↗

PubMed1981

Regulation of DNA replication: "target" determinant of the replication control elements of plasmid R6-5 lies within a control element gene.

The replication control system of plasmid R6-5 has been investigated by characterization of high-copy-number mutant miniplasmids, development of an in vivo assay for the site of action or "target" of the replication control elements, and sequence analysis of the replication control regions of the wild-type plasmid and two copy-number mutant derivatives. These and other experiments have shown that three plasmid determinants--copA/incA, copB, and copT--are involved in DNA replication control. The products of the copB and copA/incA genes, a 9500-dalton basic polypeptide and either a 7200-dalton basic polypeptide or a short untranslated RNA molecule, respectively, are negative-acting elements that interact with the third element, their target, the copT DNA sequence, or its product to regulate the frequency of initiation of plasmid replication. The location of copT within the copA/incA gene and 1600 base pairs upstream from the origin of replication indicates that regulation is effected at a preinitiation stage of replication, such as the production of a primer or other initiation factor.

Base Sequence↗

PubMed1982

Protein complexes from active replicative fractions associate in vitro with the replication origins of yeast 2-micrometers DNA plasmid.

In a search for a replication complex, the activity that replicates the 2-micrometers yeast DNA plasmid in vitro was isolated in a high molecular weight form (Mr approximately 2 X 10(6) by gel filtration and rate-zonal sedimentation from extracts prepared from cells of the budding yeast Saccharomyces. When obtained from cells in late logarithmic cultures this material or "complex" was labile compared to that from early logarithmic cultures, and it did not survive as a complex after ammonium sulfate precipitation. This suggests that, as cultures approach stationary phase and cells cease growth, the association of its protein constituents may be altered. A chimera of 2-micrometers DNA inserted into the plasmid pBR322 was used to test for binding of components of the complex. After a brief incubation of the chimera in vitro with the high molecular weight material containing replicating activity, a protein "knob" was found associated with the 2-micrometers DNA as shown by electron microscopy. This association was not random but was limited to two positions on the plasmid. In the same series of experiments, the in vitro origins of 2-micrometers plasmid replication were also mapped. Two origins were found, consistent in position with those that have been identified in vivo. Molecules utilizing both origins simultaneously in vitro were not observed, and replication in vitro was bidirectional. The location of the origins corresponded to the positions at which the protein knobs associated with 2-micrometers DNA. This and the fact that no replicative intermediates with associated complexes were detected raises the possibility that a specific protein complex may be involved in initiation of DNA replication.

DNA Replication↗

PubMed1998

Formation of a DNA loop at the replication fork generated by bacteriophage T7 replication proteins.

Intermediates in the replication of circular and linear M13 double-stranded DNA by bacteriophage T7 proteins have been examined by electron microscopy. Synthesis generated double-stranded DNA molecules containing a single replication fork with a linear duplex tail. A complex presumably consisting of T7 DNA polymerase and gene 4 helicase/primase molecules was present at the fork together with a variable amount of single-stranded DNA sequestered by gene 2.5 single-stranded DNA binding protein. Analysis of the length distribution of Okazaki fragments formed at different helicase/primase concentrations was consistent with coupling of leading and lagging strand replication. Fifteen to forty percent of the templates engaged in replication have a DNA loop at the replication fork. The loops are fully double-stranded with an average length of approximately 1 kilobase. Labeling with biotinylated dCTP showed that the loops consist of newly synthesized DNA, and synchronization experiments using a linear template with a G-less cassette demonstrated that the loops are formed by active displacement of the lagging strand. A long standing feature of models for coupled leading/lagging strand replication has been the presence of a DNA loop at the replication fork. This study provides the first direct demonstration of such loops.

Bacteriophage M13↗