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At least 973 records · Page 54Linked to original sources
PubMed1996

Smooth muscle DNA replication in response to angiotensin II is regulated differently in the neointima and media at different times after balloon injury in the rat carotid artery. Role of AT1 receptor expression.

We have reported that angiotensin II (Ang II) infusion to rats during the third and fourth weeks after vascular injury stimulates DNA replication in a larger proportion of smooth muscle cells (SMCs) in the arterial neointima than in the underlying media or the normal arterial media. Whether this increased responsiveness to Ang II is a transient or stable property of neointimal cells after vascular injury remained unclear. The present study examined smooth muscle DNA replication in response to Ang II infusion (250 ng.kg-1.min-1 for 2 weeks) at 3 to 4, 9 to 10, or 27 to 28 weeks after balloon injury to the rat carotid artery. Control rats received Ringer's lactate. BrdU (0.8 mg.kg-1.d-1) was coinfused to label replicating DNA. The increased replicative response to Ang II in the neointima versus the normal arterial media did not persist beyond the period of rapid lesion growth shortly after injury, even in neointimal areas without endothelial regeneration. By 9 to 10 weeks after injury, replication frequencies were comparable in the neointima and the normal arterial wall. In the presence of a regenerated endothelium, neointimal DNA replication was lowered but not abolished. After the early period, however, the most marked difference may be the loss of ability of medial SMCs to respond mitogenically to systemic Ang II. As a consequence, Ang II-induced DNA replication in injured arteries was greater in the neointima than in the underlying media at all times studied after injury. DNA replication levels correlated with AT1 receptor levels in the injured artery neointima but not media, as shown by receptor binding in vascular sections at 3 and 10 weeks after injury. The growth response to systemic Ang II is differentially regulated in adjacent smooth muscle layers in the injured arterial wall in vivo via mechanisms that include, but are not restricted to, the regulation of AT1 receptor expression in SMCs.

Angiotensin II↗
PubMed1985

Glucose-stimulated DNA replication of the pancreatic islets during the development of the rat fetus. Effects of nutrients, growth hormone, and triiodothyronine.

DNA replication and insulin release have been studied in islets isolated, using a tissue culture technique, from rat fetuses of different gestational ages. The islets were cultured for 3 days in media with high and low concentrations of glucose or amino acids. The DNA replication was determined by autoradiography and the insulin secreted into the medium was measured by radioimmunoassay. In islets of 22-day-old fetuses, DNA replication was stimulated by both glucose and amino acids. At gestational days 18 and 20, only amino acids increased DNA replication. However, both high glucose and high amino acid concentrations increased the islet insulin secretion into the culture medium at all ages studied. In an attempt to induce glucose-sensitive DNA replication in vitro, islets obtained from 18- and 20-day-old fetal pancreata were cultured in the presence of either triiodothyronine or human growth hormone. Triiodothyronine failed to influence either DNA replication or insulin release. Growth hormone, however, increased DNA replication and insulin release in both the experimental groups but did not induce a growth response to glucose. It is concluded that the appearance of glucose-stimulated B-cell growth is a late event in the fetal development of the rat, parallelling the late maturation of both insulin biosynthesis and release. This finding may explain the difficulties in producing islet cell hyperplasia and diabetic fetopathy previously shown in rat models of diabetes in pregnancy.

Amino Acids↗
PubMed2000

Species specificity of human RPA in simian virus 40 DNA replication lies in T-antigen-dependent RNA primer synthesis.

Replication protein A (RPA) is a three-subunit protein complex with multiple functions in DNA replication. Previous study indicated that human RPA (h-RPA) could not be replaced by Schizosaccharomyces pombe RPA (sp-RPA) in simian virus 40 (SV40) replication, suggesting that h-RPA may have a specific function in SV40 DNA replication. To understand the specificity of h-RPA in replication, we prepared heterologous RPAs containing the mixture of human and S.pombe subunits and compared these preparations for various enzymatic activities. Heterologous RPAs containing two human subunits supported SV40 DNA replication, whereas those containing only one human subunit poorly supported DNA replication, suggesting that RPA complex requires at least two human subunits to support its function in SV40 DNA replication. All heterologous RPAs effectively supported single-stranded (ss)DNA binding activity and an elongation of a primed DNA template catalyzed by DNA polymerase (pol) alpha and delta. A strong correlation between SV40 DNA replication activity and large tumor antigen (T-ag)-dependent RNA primer synthesis by pol alpha-primase complex was observed among the heterologous RPAs. Furthermore, T-ag showed a strong interaction with 70- and 34-kDa subunits from human, but poorly interacted with their S.pombe counterparts, indicating that the specificity of h-RPA is due to its role in RNA primer synthesis. In the SV40 replication reaction, the addition of increasing amounts of sp-RPA in the presence of fixed amount of h-RPA significantly reduced overall DNA synthesis, but increased the size of lagging strand, supporting a specific role for h-RPA in RNA primer synthesis. Together, these results suggest that the specificity of h-RPA in SV40 replication lies in T-ag-dependent RNA primer synthesis.

Antigens, Polyomavirus Transforming↗
PubMed1969

The negative control mechanism for E. coli DNA replication.

Evidence is presented to show that the initiation of DNA replication in E. coli 555-7 requires synthesis of a protein whose production is correlated with total protein synthesis. Once replication is initiated, however, reinitiation will occur if all further protein synthesis is prevented; a small amount of protein synthesis is sufficient to prevent this unregulated reinitiation. This shows that the initiation of DNA replication is under negative control. A mechanism for the control of DNA replication is proposed; in this mechanism a replication repressor is synthesized periodically, while an antirepressor protein is synthesized continuously. Derepression of initiation results after sufficient accumulation of the antirepressor protein, and repression is re-established by repressor synthesis after the initiation of replication.

Bacterial Proteins↗
PubMed1990

Characterization of a DNA binding protein of bacteriophage PRD1 involved in DNA replication.

Escherichia coli phage PRD1 protein P12, involved in PRD1 DNA replication in vivo, has been highly purified from E. coli cells harbouring a gene XII-containing plasmid. Protein P12 binds to single-stranded DNA as shown by gel retardation assays and nuclease protection experiments. Binding of protein P12 to single-stranded DNA increases about 14% the contour length of the DNA as revealed by electron microscopy. Binding to single-stranded DNA seems to be cooperative, and it is not sequence specific. Protein P12 also binds to double-stranded DNA although with an affinity 10 times lower than to single-stranded DNA. Using the in vitro phage phi 29 DNA replication system, it is shown that protein P12 stimulates the overall phi 29 DNA replication.

Coliphages↗
PubMed2003

An ATR- and Cdc7-dependent DNA damage checkpoint that inhibits initiation of DNA replication.

We have analyzed how single-strand DNA gaps affect DNA replication in Xenopus egg extracts. DNA lesions generated by etoposide, a DNA topoisomerase II inhibitor, or by exonuclease treatment activate a DNA damage checkpoint that blocks initiation of plasmid and chromosomal DNA replication. The checkpoint is abrogated by caffeine and requires ATR, but not ATM, protein kinase. The block to DNA synthesis is due to inhibition of Cdc7/Dbf4 protein kinase activity and the subsequent failure of Cdc45 to bind to chromatin. The checkpoint does not require pre-RC assembly but requires loading of the single-strand binding protein, RPA, on chromatin. This is the biochemical demonstration of a DNA damage checkpoint that targets Cdc7/Dbf4 protein kinase.

Animals↗
PubMed2002

Kinetic model of DNA replication in eukaryotic organisms.

We formulate a kinetic model of DNA replication that quantitatively describes recent results on DNA replication in the in vitro system of Xenopus laevis prior to the mid-blastula transition. The model describes well a large amount of different data within a simple theoretical framework. This allows one, for the first time, to determine the parameters governing the DNA replication program in a eukaryote on a genome-wide basis. In particular, we have determined the frequency of origin activation in time and space during the cell cycle. Although we focus on a specific stage of development, this model can easily be adapted to describe replication in many other organisms, including budding yeast.

Animals↗
PubMed1994

Targeting of DNA polymerase to the adenovirus origin of DNA replication by interaction with nuclear factor I.

Efficient initiation by the DNA polymerase of adenovirus type 2 requires nuclear factor I (NFI), a cellular sequence-specific transcription factor. Three functions of NFI--dimerization, DNA binding, and activation of DNA replication--are colocalized within the N-terminal portion of the protein. To define more precisely the role of NFI in viral DNA replication, a series of site-directed mutations within the N-terminal domain have been generated, thus allowing the separation of all three functions contained within this region. Impairment of the dimerization function prevents sequence-specific DNA binding and in turn abolishes the NFI-mediated activation of DNA replication. NFI DNA-binding activity, although necessary, is not sufficient to activate the initiation of adenovirus replication. A distinct class of NFI mutations that abolish the recruitment of the viral DNA polymerase to the origin also prevent the activation of replication. Thus, a direct interaction of NFI with the viral DNA polymerase complex is required to form a stable and active preinitiation complex on the origin and is responsible for the activation of replication by NFI.

Adenoviruses, Human↗
PubMed1974

Mechanism of DNA replication in Drosophila chromosomes: structure of replication forks and evidence for bidirectionality.

The replicating chromosomal DNA in Drosophila melanogaster cleavage nuclei has been visualized in the electron microscope as a serial array of closely spaced replicated regions created by pairs of diverging replication forks. The fine structure of the forks is very similar to that observed for the replication forks of bidirectionally replicating bacteriophage DNAs. However, the mean length of the single-stranded gaps in Drosophila forks is less than 200 nucleotide residues, much shorter than the gaps in phage forks. This difference in gap length corresponds to the observed difference in the size of Okazaki fragments from Drosophila and phage.

Animals↗
PubMed2000

Structure of simian virus 40 DNA replicated by herpes simplex virus type 1.

Replicating herpes simplex virus type 1 (HSV-1) DNA is known to form large branched structures. The aim of this study was to define whether HSV-1-specific DNA elements in cis play a critical role in formation of this structure. We did this by investigating the structure of heterologous simian virus 40 (SV40) DNA, which is replicated in HSV-infected cells by SV40 large T-antigen and defined HSV-encoded replication factors (e.g., DNA polymerase, single-stranded DNA-binding protein, and helicase-primase). During this process, extrachromosomal concatemeric DNA replication products are formed, indicating a herpesvirus-specific replication mode. In this study, we found that the replicating SV40 DNA consisted of a complex branched structure indistinguishable from that of replicating HSV DNA. Thus, no HSV-specific DNA element is necessary in cis for the formation of the large branched structure during HSV DNA replication. The trans-acting HSV DNA replication proteins seem to be sufficient to generate these complex structures. Moreover, replicating SV40 DNA showed a high frequency of homologous recombination events, which is typical for HSV DNA replication. However, in contrast to HSV origin-bearing amplicon plasmids, SV40 plasmids bearing the HSV cleavage-packaging signal were not efficiently processed to linear 150-kb DNA packaged into HSV capsids. This indicates that initiation of DNA synthesis on HSV-ori determines some, yet undefined, property of replicating HSV DNA, which is crucial for regular processing of the replication intermediates to daughter genomes.

Animals↗
PubMed2002

DNA replication is required for the checkpoint response to damaged DNA in Xenopus egg extracts.

Alkylating agents, such as methyl methanesulfonate (MMS), damage DNA and activate the DNA damage checkpoint. Although many of the checkpoint proteins that transduce damage signals have been identified and characterized, the mechanism that senses the damage and activates the checkpoint is not yet understood. To address this issue for alkylation damage, we have reconstituted the checkpoint response to MMS in Xenopus egg extracts. Using four different indicators for checkpoint activation (delay on entrance into mitosis, slowing of DNA replication, phosphorylation of the Chk1 protein, and physical association of the Rad17 checkpoint protein with damaged DNA), we report that MMS-induced checkpoint activation is dependent upon entrance into S phase. Additionally, we show that the replication of damaged double-stranded DNA, and not replication of damaged single-stranded DNA, is the molecular event that activates the checkpoint. Therefore, these data provide direct evidence that replication forks are an obligate intermediate in the activation of the DNA damage checkpoint.

Alkylation↗
PubMed2004

Identification of temperature-sensitive dnaD mutants of Staphylococcus aureus that are defective in chromosomal DNA replication.

The DnaD protein in Gram-positive bacteria is thought to be essential for the initiation step in DNA replication. In the present study, we characterized two Staphylococcus aureus mutants whose temperature-sensitive growth phenotype could be complemented by a plasmid carrying the dnaD gene. These mutants each had a single amino acid substitution in the DnaD protein and showed decreased DNA synthesis at restrictive temperature. Analyses of the origin to terminus ratio by Southern blotting, and of origin numbers per cell by flow cytometry, revealed that, at the restrictive temperature, one mutant continued ongoing DNA replication but failed to initiate DNA replication. The other mutant, in contrast, could not complete ongoing DNA replication and proceeded to degrade the chromosome. However, if protein synthesis was inhibited, the second mutant could complete DNA replication. These results suggest that DnaD protein is necessary not only for the initiation step, but also to avoid replication fork blockage. Moreover, both mutants were sensitive to mitomycin C, a drug that induces DNA damage, suggesting that the DnaD protein is also involved in DNA repair.

Amino Acid Substitution↗
PubMed1985

Cell lethality after selective irradiation of the DNA replication fork.

It has been suggested that nascent DNA located at the DNA replication fork may exhibit enhanced sensitivity to radiation damage. To evaluate this hypothesis, Chinese hamster ovary cells (CHO) were labeled with 125I-iododeoxyuridine (125IUdR) either in the presence or absence of aphidicolin. Aphidicolin (5 micrograms/ml) reduced cellular 125IUdR incorporation to 3-5% of the control value. The residual 125I incorporation appeared to be restricted to low molecular weight (sub-replicon sized) fragments of DNA which were more sensitive to micrococcal nuclease attack and less sensitive to high salt DNase I digestion than randomly labeled DNA. These findings suggest that DNA replicated in the presence of aphidicolin remains localized at the replication fork adjacent to the nuclear matrix. Based on these observations an attempt was made to compare the lethal consequences of 125I decays at the replication fork to that of 125I decays randomly distributed over the entire genome. Regardless of the distribution of decay events, all treatment groups exhibited identical dose-response curves (D0: 101 125I decays/cell). Since differential irradiation of the replication complex did not result in enhanced cell lethality, it can be concluded that neither the nascent DNA nor the protein components (replicative enzymes, nuclear protein matrix) associated with the DNA replication site constitute key radiosensitive targets within the cellular genome.

Animals↗
PubMed1980

Conversion of partially single-stranded replicating DNA to double-stranded DNA is delayed in megaloblastic anaemia.

DNA from phytohaemagglutinin-stimulated lymphocytes which had been pulse-labelled for 1 min with [3H]deoxycytidine eluted as partially single-stranded DNA from columns of benzoylated napthoylated DEAE-cellulose. The label was transferred progressively into the double-stranded DNA fraction upon incubation in the presence of unlabelled deoxycytidine. The rate of transfer was slower in untreated lymphocytes from patients with megaloblastic anaemia than in corresponding control cells. A similar delay was also observed in normal lymphocytes treated with methotrexate or hydroxyurea. A close temporal correlation between the joining of Okazaki pieces (measured by alkaline sucrose gradients) and the transfer of the pulse label to double-stranded DNA suggested that the latter process represented the filling of gaps between Okazaki pieces. We suggest that this gap-filling step is retarded in megaloblastic anaemia and in cells treated with methotrexate or hydroxyurea.

Anemia, Macrocytic↗