Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “replication”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

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

At least 1,333 records · Page 74Linked to original sources
PubMed2001

Different regions of primase subunit p48 control mouse polyomavirus and simian virus 40 DNA replication in vitro.

DNA polymerase alpha-primase (pol-prim), a complex consisting of four subunits, is the major species-specific factor for mouse polyomavirus (PyV) and simian virus 40 (SV40) DNA replication. Although p48 is the most conserved subunit of pol-prim, it is required for in vitro PyV DNA replication but can inhibit cell-free SV40 DNA replication. Production of chimeric human-mouse p48 revealed that different regions of p48 are involved in supporting PyV DNA replication and inhibiting SV40 DNA replication. The N and C-terminal parts of p48 do not have species-specific functions in cell-free PyV DNA replication, but the central part (amino acids [aa] 129 to 320) controls PyV DNA replication in vitro. However, PyV T antigen physically binds to mouse, human, and chimeric pol-prim complexes independently, whether they support PyV DNA replication or not. In contrast to the PyV system, the inhibitory effects of mouse p48 on SV40 DNA replication are mediated by N- and C-terminal regions of p48. Thus, a chimeric p48 containing human aa 1 to 128, mouse aa 129 to 320, and human aa 321 to 418 is active in both PyV and SV40 DNA replication in vitro.

Animals↗
PubMed2002

Analysis of an Autographa californica nucleopolyhedrovirus lef-11 knockout: LEF-11 is essential for viral DNA replication.

The Autographa californica nucleopolyhedrovirus (AcMNPV) lef-11 gene was previously identified by transient late expression assays as a gene important for viral late gene expression. The lef-11 gene was not previously identified as necessary for DNA replication in transient origin-dependent plasmid DNA replication assays. To examine the role of lef-11 in the context of the infection cycle, we generated a deletion of the lef-11 gene by recombination in an AcMNPV genome propagated as a BACmid in Escherichia coli. The resulting AcMNPV lef-11-null BACmid (vAc(lef11KO)) was unable to propagate in cell culture, although a "repair" AcMNPV BACmid (vAc(lef11KO-REP)), which was generated by transposition of the lef-11 gene into the polyhedrin locus of the vAc(lef11KO) BACmid, was able to replicate in a manner similar to wild-type or control AcMNPV viruses. Thus, the lef-11 gene is essential for viral replication in Sf9 cells. The vAc(lef11KO) BACmid was examined to determine if the defect in viral replication resulted from a defect in DNA replication or from a defect in late transcription. The lef-11-null BACmid and control BACmids were transfected into Sf9 cells, and viral DNA replication was monitored. The viral DNA genome of the lef-11-null BACmid (vAc(lef11KO)) was not amplified, whereas replication and amplification of the genomes of the repair BACmid (vAc(lef11KO-REP)), wild-type AcMNPV, and a nonpropagating gp64-null control BACmid (vAc(GUSgp64KO)) were readily detected. Northern blot analysis of transcripts from selected early, late, and very late genes showed that late and very late transcription was absent in cells transfected with the lef-11-null BACmid. Thus, in contrast to prior studies using transient replication and late expression assays, studies of a lef-11-null BACmid indicate that LEF-11 is required for viral DNA replication during the infection cycle.

Animals↗
PubMed1994

DNA replication from initiation zones of mammalian cells in a model system.

We reported that DNA replication initiates from the region containing an autonomously replicating sequence from Saccharomyces cerevisiae when negatively supercoiled plasmid DNA is incubated with the proteins required for simian virus 40 DNA replication (Y. Ishimi and K. Matsumoto, Proc. Natl. Acad. Sci. USA 90:5399-5403, 1993). In this study, the DNAs containing initiation zones from mammalian cells were replicated in this model system. When negatively supercoiled DNA containing an initiation zone (2 kb) upstream of the human c-myc gene was incubated with simian virus 40 T antigen as a DNA helicase, HSSB (also called replication protein A), and DNA polymerase alpha-primase complex isolated from HeLa cells, DNA replication was specifically initiated from the center of the initiation zone, which was elongated bidirectionally in the presence of a DNA swivelase. Without HSSB, the level of DNA synthesis was significantly reduced and the localized initiation could not be detected, indicating that HSSB plays an essential role in the initiation of DNA replication. The digestion of negatively supercoiled template DNA with a single-strand-specific nuclease revealed that HSSB stimulated DNA unwinding in the center of the initiation zone where the DNA duplex is relatively unstable. In contrast, DNA replication started from a broad region of an initiation zone downstream of the dihydrofolate reductase gene from chinese hamster ovary cells, but the center of the region was mapped near the origin of bidirectional DNA replication. These results suggested that this system mimics a fundamental process of initiation of eukaryotic DNA replication. The mechanism of initiation is discussed.

Base Sequence↗
PubMed1988

Functional analysis of the individual enhancer core sequences of polyomavirus: cell-specific uncoupling of DNA replication from transcription.

Polyomavirus (Py) enhancer core elements were compared for their ability to activate Py early transcription and DNA replication in mouse 3T6 cells, lymphoid cell lines, and undifferentiated embryonal carcinoma cells. By examining the pattern of genetic change in a number of cell-specific Py variants, we identified subenhancer sequences that may be functionally important for virus replication. Four such distinct enhancer consensus sequences were synthesized and designated as the A core (homologous with adenovirus 5 E1A enhancer), B core (homologous to the simian virus 40 A enhancer core), C core (containing an inverted repeat within the Py B enhancer), and BPV core (homologous to the bovine papillomavirus enhancer). When used to replace the complete Py B enhancer, single copies of all but the BPV element were able to fully activate Py DNA replication after transfection, but this activation was usually cell type specific. In the PCC4 embryonal carcinoma cells, only the A-core sequence was able to activate transcription and DNA replication. The BPV core sequence containing the Py F441 point change was unable to activate DNA replication in the F9 embryonal carcinoma or any other cell line. No single insertion element was dominant nor did these elements display the wild-type enhancer pattern of cell-specific activation of DNA replication. In addition, differential effects were often observed on the activation of transcription versus DNA replication. In 3T6 cells, transcription could be highly activated by the A core without a corresponding activation of DNA replication. In murine T lymphoid cell lines, the B core activated DNA replication without a corresponding increase in transcription. Furthermore, both DNA replication and, to a lesser degree, transcription often showed a strong tissue-specific dependence on the polarity of the inserted core element for activation.

Animals↗
PubMed2000

DNA replication in the third domain (of life).

DNA replication is the process underlying evolution and the propagation of living organisms. Since the discovery of DNA-dependent DNA polymerases more than 40 years ago, the mechanisms governing DNA replication have been extensively studied in bacteria and eukarya. During the last several years, these studies have been extended to the third domain of life, the archaea. Although archaea are prokaryotes, their replication machinery and the proteins participating in the initiation of DNA replication are more similar to those found in eukarya than bacteria. It appears, however, that replication in archaea is a simpler version of the eukaryotic one as fewer polypeptides participate in each phase of the replication process. The archaeal replication apparatus also has several unique features not found in eukaryotic organisms. Furthermore, like bacteria, members of this domain thrive under a broad range of environmental conditions including extreme temperature, high salt, pH, etc. Thus, the replication machinery had to adapt to these extreme conditions. This article summarizes our current understanding of the mechanisms governing DNA replication in archaea and highlights similarities and differences between archaeal replication and that of bacteria and eukarya.

Archaea↗
PubMed1999

Transcription factors in DNA replication.

Accumulating evidence suggests the involvement of transcription factors in the regulation of DNA replication in eukaryotic cells. Almost all eukaryotic DNA viruses contain binding sites for transcription factors which function as auxiliary elements for DNA replication initiation at replication origins, and, indeed, the binding of transcription factors to these elements has been shown to stimulate DNA replication. Transcription factors also regulate some of the chromosome DNA replication origins of budding yeast, indicating that transcription factor involvement in DNA replication is not restricted to viruses. Consistent with this notion, recently determined replication origins of higher eukaryotes have been found occasionally to associate with transcription factor binding sites, although there is no direct evidence for the involvement of the factors that bind to these sequences in DNA replication. Analyses using viral and yeast systems have suggested that transcription factors stimulate the formation of the replication initiation complex by engaging in specific interactions with proteins of the initiation complex and/or by modulating the repressive chromatin structure around origins of replication. These mechanisms are analogous to those advanced to explain stimulation of transcription by transcription factors. The accumulated data suggests that transcription factors play a general role in the formation of functional complexes on chromosomes.

Animals↗
PubMed2002

Enzyme-activated Prodrug Therapy Enhances Tumor-specific Replication of Adenovirus Vectors.

Adenoviruses (Ads) that selectively replicate in tumor cells have shown promising preliminary results in clinical trials, especially in combination with chemotherapy. Here, we describe a system that combines the antitumor synergy of Ads and chemotherapeutic agents with the benefits of enzyme-activated prodrug therapy. In this system, a functional transgene expression cassette is created by homologous recombination during adenoviral DNA replication. Transgene expression is strictly dependent on viral DNA replication, which in turn is tumor specific. We constructed replication-activated Ad vectors to express a secreted form of beta-glucuronidase and a cytosine deaminase/uracil phosphoribosyltransferase, which activate the prodrugs 9-aminocamptothecin glucuronide to 9-aminocamptothecin and 5-fluorocytosine to 5-fluorouracil (5-FU) and further to 5-fluoro-UMP, respectively. We demonstrated replication-dependent transgene expression, prodrug activation, and induction of tumor cell toxicity by secreted beta-glucuronidase and cytosine deaminase/uracil phosphoribosyltransferase. Furthermore, exposure of cells to activated prodrug or drug at subtoxic concentrations enhanced viral DNA replication. Characteristically, these agents induced changes in the cell cycle status of exposed cells (G(2) arrest), which closely resembled the effect of wild-type Ad infection, and are thought to be favorable for viral replication. We tested a number of cytostatic drugs (camptothecin, etoposide, daunorubicin, cisplatin, 5-fluorouracil, hydroxyurea, Taxol, and actinomycin D) for their effect on viral DNA replication and found considerable differences between individual agents. Finally, we show that the combination of viral and prodrug therapy enhances viral replication and spread in liver metastases derived from human colon carcinoma or cervical carcinoma in a mouse model. Our data indicate that specific vector/drug combinations tailored to be synergistic may have the potential to improve the potency of either therapeutic approach. These data also provide a new rationale for expressing prodrug-activating enzymes from conditionally replicating Ads.

Adenoviridae↗
PubMedDate not supplied

[The eukaryotic replication complex and its affinity modification analysis].

Replication of eukaryotic DNA is driven by a protein complex, in which the central part is played by DNA polymerases. Synthesis with eukaryotic DNA polymerases alpha, delta, and epsilon involves various replication factors, including the replication protein A, replication factor C, proliferating cell nuclear antigen, etc. Replication enzymes and factors also participate in DNA repair, which is in an interplay with DNA replication. The function of the entire multicomponent system is regulated by protein--nucleic acid and protein--protein interactions. The eukaryotic replication complex was not isolated as a stable supramolecular structure, suggesting its dynamic organization. Hence X-ray analysis and other instrumental techniques are hardly suitable for studying this system. An alternative approach is affinity modification. Its most promising version involves in situ generation of photoreactive DNA replication intermediates. The review considers the recent progress in photoaffinity modification studies of DNA polymerases, eukaryotic replication factors, and their interactions with DNA replication intermediates.

Affinity Labels↗
PubMed1996

Application of an in vitro system in the study of chemotherapeutic drug effects on DNA replication.

DNA replication machinery is an important target for chemotherapeutic drugs. We have used an in vitro system to study the effect of drugs on mammalian DNA replication, either by direct interaction with the DNA structure or with replication proteins and machinery. The anthracycline doxorubicin (Dox) showed a dose-dependent inhibitory effect on DNA replication, whether incubated with HeLa cell extracts or with DNA and nucleotides. Earliest-labeled fragment analysis revealed that inhibition of replication began within the origin-containing fragment in both control and Dox-containing reactions in vitro. AraC, a nucleoside analog, had no significant effect on DNA synthesis. In contrast, araCTP was able to inhibit DNA replication in vitro. Since metabolism is diminished in this in vitro system, the degree of phosphorylation of araC was apparently low. Progesterone showed an increase in nucleotide incorporation (sensitive to BuPdGTP inhibition of replication-specific polymerases alpha and delta) after preincubation with HeLa cell extracts, although progesterone receptors were not detectable in the HeLa cell extracts. In addition, we observed an inhibition in DNA replication when progesterone was preincubated with DNA and nucleotides. These results suggest that progesterone may have a mechanism of action that is different from any known to be mediated through progesterone receptors. In conclusion, these results indicate that this mammalian in vitro replication system will be useful for the study of mechanisms and design of therapeutic drugs that inhibit mammalian DNA replication.

Antibiotics, Antineoplastic↗
PubMed1978

Replication patterns of three isodicentric X chromosomes and an X isochromosome in human lymphocytes.

Chromosomes from four patients with variants of the Turner syndrome were investigated by G- and C-bandind and DNA replication techniques. Their karyotypes were: 1) 46,X,idic(X)(q28), 2) 45,X/46,X,idic(X)(q24), 3) 45,X/46,X,idic(X)(p11), and 4) 46,X,i(Xq). In patients 1, 2, and 3, the abnormal X was isodicentric, with different break-and-fusion points in each case. In each, the G-band pattern on one side of the breakpoint was a mirror image of that on the other side. Each had two distinct C-bands, only one of which was associated with a primary constriction. The fourth patient had an isochromosome of the long arm of an X in which only one C-band could be discerned. Replication studies were done on lymphocyte cultures by incorporating a thymidine analogue and staining with acridine orange. In addition, replication patterns of normal early- and late-replicating X chromosomes were studied in two normal females. In the four patients, all the normal X chromosomes had normal early-replication patterns. The two idic(X) chromosomes with break-and-fusion points on their long arms almost always had symmetric replication patterns, which demonstrates that the corresponding bands replicated synchronously. In contrast, many of the idic(X)(p11) and i(Xq) chromosomes showed asymmetric or asynchronous replication. In each, the replication pattern of the abnormal X was similar to the equivalent portions of a normal late-replicating X.

Adolescent↗
PubMed1983

Specific terminal DNA replication sequence of X chromosomes in different tissues of a live-born triploid infant.

Using the thymidine pulse method, DNA replication kinetics were studied on cells derived from cartilage, gonad, lymphocytes, and skin of a live-born triploid (69,XXY) infant with typical clinical findings. Replication studies showed that 3% of the lymphocytes had one early and one late replicating X, and 97% of the lymphocytes, and cartilage, gonad, and skin cells had two early replicating X's. Asynchronous DNA replication between the two early replicating X's was observed in all tissues (range 25-40%). The predominant terminal replication sequence of X chromosomes from chondrocytes, gonad, and skin fibroblast differed from that of the lymphocytes. Thus, a tissue-specific DNA replication pattern of the early-replicating X chromosome may be present. In every tissue, the last band to complete DNA replication was Xq21. Polymorphisms of metaphase chromosomes of parents and the patient were studied by Q-banding. The possible origin of the extra haploid set of chromosomes is discussed.

DNA Replication↗
PubMed1991

DNA replication in cell-free extracts from Drosophila melanogaster.

We have developed an efficient in vitro replication system from 0-2 h Drosophila melanogaster embryos. Demembranated Xenopus sperm DNA when incubated in such an extract first becomes enclosed in a nucleus-like structure with a nuclear envelope and a karyoskeleton. It then undergoes one round of semiconservative replication--this replication appears completely dependent on nuclear formation. Up to 30% of input DNA is nucleated in one reaction. Efficient nuclear formation and replication are dependent on a cold treatment step, prior to disruption of the embryos. They also depend on the age of the embryos used. Extracts from older embryos (0-5 h) are capable of nuclear formation, although at a much reduced efficiency, and repair synthesis, but seem to have lost the ability to initiate DNA replication. In addition to replicating sperm DNA this system appears capable of carrying out semi-conservative replication on some plasmids. However, it cannot use these to trigger nuclear formation; replication is only seen if the plasmids are coincubated with sperm DNA. The in vitro formed nuclei have not been observed to trigger nuclear envelope breakdown and entry into mitosis. However, they can re-replicate the DNA if the nuclei are permeabilized. This system should be a useful complement to the previously isolated Xenopus in vitro replication system. In addition the amenability of Drosophila to genetic study should open up new approaches not previously possible with Xenopus.

Animals↗
PubMed1994

Replicator dominance in a eukaryotic chromosome.

Replicators are genetic elements that control initiation at an origin of DNA replication (ori). They were first identified in the yeast Saccharomyces cerevisiae as autonomously replicating sequences (ARSs) that confer on a plasmid the ability to replicate in the S phase of the cell cycle. The DNA sequences required for ARS function on a plasmid have been defined, but because many sequences that participate in ARS activity are not components of chromosomal replicators, a mutational analysis of the ARS1 replicator located on chromosome IV of S. cerevisiae was performed. The results of this analysis indicate that four DNA elements (A, B1, B2 and B3) are either essential or important for ori activation in the chromosome. In a yeast strain containing two closely spaced and identical copies of the ARS1 replicator in the chromosome, only one is active. The mechanism of replicator repression requires the essential A element of the active replicator. This element is the binding site for the origin recognition complex (ORC), a putative initiator protein. The process that determines which replicator is used, however, depends entirely upon flanking DNA sequences.

Chromosomes, Fungal↗
PubMed1994

The mechanism of the stringent control of lambda plasmid DNA replication.

Lambda plasmid DNA replication is inhibited in amino acid-starved wild type Escherichia coli strains (stringent response) but not in amino acid-starved relA mutants (relaxed response). This replication is perpetuated by the replication complex containing the lambda O protein (which is protected from proteases by other elements of the complex) and inherited by one of two daughter copies after a replication round. Since a fraction of stable lambda O protein was observed in relA- and relA+ strains, and negative regulation by the lambda Cro repressor does not seem to be important in the stringent or relaxed response of lambda plasmid replication to amino acid starvation, the inhibition of lambda plasmid replication in amino acid-starved wild type strains was investigated. lambda plasmids were unable to replicate in amino acid-starved relA- bacteria treated with rifampicin. Moreover, transcription from pR, which produces mRNA for replication protein synthesis and serves as transcriptional activation of ori lambda, was significantly decreased during the stringent response as well as in non-starved cells containing increased levels of ppGpp. However, it was little or totally not affected by the relaxed response. The replacement of pR with plac (which is known to be uninhibited by ppGpp) in a lambda plasmid resulted in its DNA replication during relaxed and stringent responses as well as during overproduction of ppGpp in unstarved bacteria. We conclude that ppGpp-mediated inhibition of transcriptional activation of ori lambda is responsible for inhibition of lambda plasmid DNA replication in amino acid-starved wild type strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage lambda↗
PubMed2006

A role of the mitotic spindle checkpoint in the cellular response to DNA replication stress.

Replication stress is a frequent and early event during tumorigenesis. Whereas the cellular responses to a persistent block of replication fork progression have been extensively studied, relatively little is known about how cells respond to low-intensity replication stress. However, transient replication fork perturbations are likely to occur even more frequently in tumor cells than a permanent replication arrest. We report here that transient, low intensity replication stress leads to a rapid activation of the DNA replication checkpoint but to a significantly delayed apoptotic response in a small but significant number of cells. This late apoptotic response was independent of p53 and we found evidence for cell death during mitosis in a proportion of cells. To further explore the role of p53 in the response to replication stress, we analyzed mouse embryonic fibroblasts (MEFs) deficient of p53 in comparison to wild-type or p63- or p73-deficient MEFs. We detected a significant increase of apoptosis and morphological signs of failed mitosis such as multinucleation in p53-deficient MEFs following replication stress, but not in wild-type or p63- or p73-deficient cells. Multinucleated p53-deficient MEFs frequently retained cyclin B1 expression indicating a persistently activated mitotic spindle checkpoint. Collectively, our results suggest that the cellular response to replication stress involves the mitotic spindle checkpoint in a proportion of cells. These findings imply that the mitotic spindle checkpoint may act in concert with DNA damage and cell-cycle checkpoints as an early anti-tumor barrier and provide a possible explanation for its frequent relaxation in human cancer.

Animals↗
PubMed1988

Timing of proto-oncogene replication: a possible determinant of early S phase sensitivity of C3H 10T1/2 cells to transformation by chemical carcinogens.

The temporal order of replication of several genes was studied in 10T1/2 cells synchronized by release from confluence-induced arrest of proliferation followed by treatment with 2 micrograms/mL aphidicolin for 24 h. DNA subjected to bromodeoxyuridine substitution for 1- or 2-h intervals spanning the S phase was separated from the remaining DNA in cesium chloride gradients, filtered onto nitrocellulose in a slot-blot apparatus, and hybridized with various 32P-labeled probes. Ha-ras was among the first genes replicated at the onset of the S phase. The myc proto-oncogene replicated later but within the first hour of the S phase. The replication of Ki-ras, raf, and mos was detected between hour 1 and 2 of the S phase. The dihydrofolate reductase gene replicated early (0-2 h) and the myb proto-oncogene replicated in mid-S phase (2-4 h). An immunoglobulin VH sequence and the beta-globin gene replicated late in 10T1/2 cells, 4-6 h after removal of aphidicolin. Replicating DNA is preferentially adducted by chemical carcinogens, and replication of damaged proto-oncogenes before they are repaired may activate their transforming potential. Therefore, the observed replication of proto-oncogenes during the early S phase may underlie the enhanced sensitivity of 10T1/2 cells to chemically induced transformation at this point in the cell cycle.

Animals↗
PubMed1994

An additional role of transcriptional activation of ori lambda in the regulation of lambda plasmid replication in Escherichia coli.

Initiation of replication of plasmids derived from coliphage lambda in vivo is dependent on transcription at or near the replication origin, ori lambda. However, this transcriptional activation is dispensable for lambda plasmid DNA replication reconstituted in vitro from purified lambda and Escherichia coli proteins. It was proposed previously that histone-like protein HU interferes with the assembly or function of the pre-primosomal complex, and transcription at or near ori lambda abolishes HU-mediated inhibition of lambda DNA replication. We found that during lambda plasmid replication driven by the previously assembled replication complex (in amino acid-starved relA mutants), when the inhibition by HU protein should not be observed, the synthesis of lambda DNA was still dependent on transcriptional activation. Moreover, in hupA hupB double mutants the transcription is necessary for the initiation of lambda plasmid DNA replication perpetuated by the replication complex inherited by one of two daughter copies after a replication round. We conclude that transcriptional activation of ori lambda has an additional role in the initiation of lambda plasmid DNA replication beside the abolition of HU-mediated inhibition.

Bacterial Proteins↗
PubMed2001

Replication timing of human telomeric DNA and other repetitive sequences analyzed by fluorescence in situ hybridization and flow cytometry.

The replication timing of telomeres seems to differ between species. Yeast telomeres are late replicating, whereas limited data from very few human cell lines have indicated telomere replication throughout S phase. In the present study a series of permanent cell lines and patient samples was investigated using a flow cytometric approach for telomere length determination based on in situ hybridization using peptide nucleic acid probes and DNA staining. This method permits selective analysis of cells in specific phases of the cell cycle without perturbation of the cell cycle machinery. The timing of replication of telomeric C(3)TA(2) and T(2)AG(3) repeats was found to differ between individual samples and could precede or be concomitant with the replication of bulk DNA. Replication of the T(2)AG(3) strand seemed to occur somewhat later than that of the C(3)TA(2) strand in some samples. (GTG)(n) and other repetitive sequences generally showed a replication pattern similar to that of the bulk of DNA with slightly individual differences, whereas centromeric DNA repeats consistently replicated within a short time frame in late S phase. The apparent variability in replication timing seen for telomeric DNA might suggest individual differences in firing of replication origins.

Cell Cycle↗