Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “DNA 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 1,117 records · Page 62Linked to original sources
PubMed1990

Nucleosome assembly in mammalian cell extracts before and after DNA replication.

Protein-free DNA in a cytosolic extract supplemented with SV40 large T-antigen (T-Ag), is assembled into chromatin structure when nuclear extract is added. This assembly was monitored by topoisomer formation, micrococcal nuclease digestion and psoralen crosslinking of the DNA. Plasmids containing SV40 sequences (ori- and ori+) were assembled into chromatin with similar efficiencies whether T-Ag was present or not. Approximately 50-80% of the number of nucleosomes in vivo could be assembled in vitro; however, the kinetics of assembly differed on replicated and unreplicated molecules. In replicative intermediates, nucleosomes were observed on both the pre-replicated and post-replicated portions. We conclude that the extent of nucleosome assembly in mammalian cell extracts is not dependent upon DNA replication, in contrast to previous suggestions. However, the highly sensitive psoralen assay revealed that DNA replication appears to facilitate precise folding of DNA in the nucleosome.

Animals↗
PubMed1993

Involvement of annexin II in DNA replication: evidence from cell-free extracts of Xenopus eggs.

Cell-free extracts of Xenopus eggs efficiently initiate and complete semiconservative DNA replication of exogenously added plasmid DNA. DNA replication in such extracts can be neutralized by a monoclonal antibody (D1/274.5) against human annexin II. Specific immunodepletion of Xenopus annexin II from the egg extracts results in loss of DNA replicative ability. Immunodepletion of annexin II does not prevent nuclear assembly, a stringent requirement for DNA synthesis on exogenous DNA in this system. Replicative ability can be restored to the immunodepleted extracts by the addition of purified human annexin II. These results demonstrate that annexin II is involved in chromosomal DNA replication and has a role in the cell cycle of higher eukaryotes.

Animals↗
PubMed1984

DNA replication of simian virus 40 chromatin in digitonin-treated and saponin-treated permeable cells.

Permeable cell systems have been developed by treatment of simian virus 40 (SV40)-infected cells with digitonin or saponin for studying in vitro DNA replication of chromatin. SV40 DNA replicated efficiently in these systems in the presence of the four deoxyribonucleoside triphosphates, ATP, Mg2+, and the proper ionic environment. Analysis of the time course of the replicated SV40 DNA I, II and replicating intermediates by gel electrophoresis and autoradiography indicated that SV40 DNA replicated completely in the form of nucleoprotein complexes (SV40 chromatin).

Animals↗
PubMed1985

RecA protein acts at the initiation of stable DNA replication in rnh mutants of Escherichia coli K-12.

Escherichia coli rnh mutants lacking RNase H activity are capable of recA+-dependent DNA replication in the absence of concomitant protein synthesis (stable DNA replication). In rnh dnaA::Tn10 and rnh delta oriC double mutants in which the dnaA+-dependent initiation of DNA replication at oriC is completely blocked, the recA200 mutation encoding a thermolabile RecA protein renders both colony formation and DNA synthesis of these mutants temperature sensitive. To determine which stage of DNA replication (initiation, elongation, or termination) was blocked, we analyzed populations of these mutant cells incubated at 30 or 42 degrees C in the presence or absence of chloramphenicol (CM) by dual-parameter (DNA-light scatter) flow cytometry. Incubation at 30 degrees C in the presence of CM resulted in cells with a continuum of DNA content up to seven or more chromosome equivalents per cell. The cultures which had been incubated at 42 degrees C in the absence or presence of CM consisted of cells with integral numbers of chromosomes per cell. It is concluded that active RecA protein is required specifically for the initiation of stable DNA replication.

Chloramphenicol↗
PubMed2004

Specification of a DNA replication origin by a transcription complex.

In early Xenopus development, transcription is repressed and DNA replication initiates at non-specific sites. Here, we show that a site-specific DNA replication origin can be induced in this context by the assembly of a transcription domain. Deletion of the promoter element abolishes site-specific initiation, and its relocalization to an ectopic site induces a new origin of replication. This process does not require active transcription, and specification of the origin occurs mainly through a decrease in non-specific initiation at sites distant from the promoter. Finally, chromatin immunoprecipitation experiments suggest that site-specific acetylation of histones favours the selection of the active DNA replication origin. We propose that the specification of active DNA replication origins occurs by secondary epigenetic events and that the programming of chromatin for transcription during development contributes to this selection in higher eukaryotes.

Acetylation↗
PubMed1992

DNA replication in cell-free extracts from Xenopus eggs is prevented by disrupting nuclear envelope function.

The lectin, wheat germ agglutinin (WGA), has previously been shown to prevent transport into the cell nucleus. This paper shows that WGA also inhibits nuclear DNA replication, under the same conditions that prevent transport. Although WGA eliminates sperm nuclear DNA replication in a cell-free extract of Xenopus eggs, DNA synthesis on a single-stranded template proceeds normally. Inhibition of nuclear DNA replication is partially reversed by addition of N-acetylglucosamine, and completely reversed by triacetylchitotriose. Sensitivity to inhibition by WGA is greatest during the nuclear assembly phase, and nuclear formation on sperm chromatin is blocked. DNA replication in preformed nuclear templates is also sensitive to WGA inhibition. I propose that WGA blocks DNA replication by preventing nuclear transport. The data presented here also indicate that, under certain circumstances, the elongation stage of DNA replication does not proceed in the absence of an intact nuclear envelope. The roles of the nuclear envelope and active nuclear transport in DNA replication are discussed.

Acetylglucosamine↗
PubMed1972

Electron microscopic studies of replicating and catenated colicin factor E1 DNA isolated from minicells (DNA replication).

Replicating and catenated DNA molecules of colicin factor E1 that were isolated from minicells were observed by electron microscopy. Two new replicating structures that have been found include: molecules that contain two untwisted replicated branches of the same length and a twisted unreplicated branch, and a twisted circular molecule with a long tail. Other molecular forms identified include circular dimers, three types of catenated dimers (open-open, open-twisted, and twisted-twisted), catenated dimers in which one of the linked molecules is replicating, catenated trimers, and a tetamer.

Centrifugation, Density Gradient↗
PubMed1993

Control of simian virus 40 DNA replication by the HeLa cell nuclear kinase casein kinase I.

The initiation of simian virus 40 (SV40) DNA replication is regulated by the phosphorylation state of the viral initiator protein, large T antigen. We describe the purification from HeLa cell nuclei of a 35-kDa serine/threonine protein kinase that phosphorylates T antigen at sites that are phosphorylated in vivo and thereby inhibits its ability to initiate SV40 DNA replication. The inhibition of both origin unwinding and DNA replication by the kinase is reversed by protein phosphatase 2A. As determined by molecular weight, substrate specificity, autophosphorylation, immunoreactivity, and limited sequence analysis, this kinase appears to be identical to casein kinase I, a ubiquitous serine/threonine protein kinase that is closely related to a yeast kinase involved in DNA metabolism. The HeLa cell phosphorylation cycle that controls the initiation of SV40 DNA replication may also play a role in cellular DNA metabolism.

Adenosine Triphosphate↗
PubMed1997

Nuclear sites of herpes simplex virus type 1 DNA replication and transcription colocalize at early times postinfection and are largely distinct from RNA processing factors.

We have visualized the intracellular localization of herpes simplex virus (HSV) type 1 replication and transcription sites in infected HeLa cells by using direct labelling methods. The number of viral transcription foci increases in a limited way; however, the number of replication sites increases in a near-exponential manner throughout infection, and both replication and transcription sites are found buried throughout the nuclear interior. Simultaneous visualization of viral transcription and replication foci shows that the two processes colocalize at early times, but at later times postinfection, there are additional sites committed solely to replication. This contrasts with the situation in adenovirus-infected cells in which, throughout replication, sites of transcription are adjacent to but do not colocalize with sites of viral DNA replication. The data for an increase in HSV transcription sites suggest an initial phase of replication of input genomes which are then transcribed. Sites of HSV replication colocalize with viral DNA replication and packaging proteins but are largely distinct from the punctate distribution of small nuclear ribonucleoprotein particles. Very high multiplicities of infection have shown an upper limit of some 18 viral transcription foci per nucleus, suggesting cellular constraints on transcription site formation. Use of virus replication mutants confirms that the labelled foci are sites of viral RNA and DNA synthesis; in the absence of viral DNA replication functions, no replication foci and only a limited number of transcription foci were present. Absence of a packaging function had no apparent effect on transcription or replication site formation, illustrating that DNA packaging is not a prerequisite for ongoing DNA synthesis. Further, the essential HSV protein IE63 is required for efficient replication site formation at later times postinfection but is not required for transcription foci formation.

Cell Nucleus↗
PubMed1985

Does capacity of DNA replication change during in vitro ageing?

We described elsewhere how a lack of change in the rate of DNA chain elongation occurred during in vitro ageing of human diploid fibroblasts. Here we further examined the rate of actual incorporation of tritiated thymidine, the center-to-center distance of replicons and the length of each phase of the cell cycle in order to extend our previous results to the other aspects of DNA replication. The results obtained showed that the rate of net DNA synthesis, the replicon size and the duration of S phase did not change during in vitro ageing. Our findings indicated that the reason why the greater part of the cell population at high population doubling levels becomes incapable of proliferating might not be the gradual decline in the ability of DNA replication. The regulation system(s) of DNA replication may alter during the period of culturing without any change in the capacities of the DNA replication machinery and, consequently, the non-cycling cells increase.

Autoradiography↗
PubMed1996

Processivity of the gene 41 DNA helicase at the bacteriophage T4 DNA replication fork.

The gene 41 protein is the DNA helicase associated with the bacteriophage T4 DNA replication fork. This protein is a major component of the primosome, being essential for coordinated leading and lagging strand DNA synthesis. Models suggest that such DNA helicases are loaded only onto DNA at origins of replication, and that they remain with the ensuing replication fork until replication is terminated. To test this idea, we have measured the extent of processivity of the 41 protein in the context of an in vitro DNA replication system composed of eight purified proteins (the gene 43, 44/62, 45, 32, 41, 59, and 61 proteins). After starting DNA replication in the presence of these proteins, we diluted the 41 helicase enough to prevent any association of new helicase molecules and analyzed the replication products. We measured an association half-life of 11 min, revealing that the 41 protein is processive enough to finish replicating the entire 169-kilobase T4 genome at the observed replication rate of approximately 400 nucleotides/s. This processivity of the 41 protein does not require the 59 protein, the protein that catalyzes 41 protein assembly onto 32 protein-covered single-stranded DNA. The stability we measure for the 41 protein as part of the replication fork is greater than estimated for it alone on single-stranded DNA. We suggest that the 41 protein interacts with the polymerase holoenzyme at the fork, both stabilizing the other protein components and being stabilized thereby.

Bacteriophage T4↗
PubMed1997

Overexpression of insulin-like growth factor (IGF)-I receptor enhances inhibition of DNA replication in mouse cells exposed to x-rays.

Previous studies from our laboratory provided evidence for the operation of signal transduction pathways involving ras, myc, and staurosporine-sensitive protein kinases in the regulation of DNA replication in irradiated cells. Because ras and myc are also involved in the signal transduction elicited in response to ligand activation of growth factor receptors, we wondered whether growth factor receptors are upstream elements in the regulation of DNA replication in irradiated cells. Here, we report on the role of insulin-like growth factor I receptor (IGF-IR) in the regulation of DNA replication in irradiated cells. We compare radiation-induced inhibition of DNA replication in BALB/c 3T3 cells with that in P6 cells. P6 cells are derived from BALB/c 3T3 cells by transfection with a vector expressing IGF-IR, leading to 30-fold overexpression. We observe a significantly stronger inhibition of DNA replication after irradiation in P6 as compared with BALB/c 3T3 cells at all doses examined. Sedimentation in alkaline sucrose gradients shows that the increased inhibition in P6 cells is due to an increased inhibition of replicon initiation, the main controlling event in DNA replication. Staurosprine at 20 nM reduces radiation-induced inhibition of DNA replication in BALB/c 3T3 cells, but has only a small effect in P6 cells. Caffeine at a concentration of 1 mM, on the other hand, removes over 60% of the inhibition in both cell lines. The results implicate IGF-IR in the regulation of DNA replication in irradiated cells, but also suggest differences between cells of different origins in the proteins involved in the regulating signal transduction pathway.

3T3 Cells↗
PubMed1992

Relationship of single-stranded DNA-binding proteins of Ehrlich ascites tumour to cell growth phase and DNA replications.

The possible involvement of SSB-proteins in DNA replication in Ehrlich ascites tumour (EAT) has been investigated. A direct relation (the computer-generated correlation coefficient was 0.9) between the SSB-proteins content in chromatin and intensity of the replicative synthesis of DNA in various preparation of EAT in vivo and in vitro is observed. Addition of exogenous SSB-proteins to the permeable EAT cells has been found to increase the replicative synthesis. Although eukaryotic SSB-proteins are not complete analogs of the prokaryotic SSB-proteins, they evidently participate in DNA replication in eukaryotic cells and possibly are intracellular regulators of proliferation.

Animals↗
PubMed1975

Physiological transition of a coliphage lambda DNA replication.

The "rolling-circle" replicative intermediate (sigma-type molecules) which is normally produced in the late stage of coliphage lambda DNA replication can be found during the first round of lambda DNA replication if cells infected with lambda replication mutant Ots28 are incubated at the nonpermissive temperature until the late stage of the latent period of lambda infection. After shifting to the permissive temperature, the vast majority of replicating forms are sigma-type rolling circle even during the first round of DNA replication. Concatemeric lambda DNA molecules, produced from these sigma-type intermediates, are efficiently packaged into progeny phage, indicating that in the first round of lambda DNA replication, double-branched theta-type molecules are not required for production of viable progeny phage.

Centrifugation, Density Gradient↗
PubMed1995

Expression of human cytomegalovirus UL36 and UL37 genes is required for viral DNA replication.

It was previously reported that the region encoding human cytomegalovirus (HCMV) genes UL36 to UL38 was required for origin-dependent DNA replication. These genes encode transactivators that upregulate viral and cellular transcription. However, their requirement for viral DNA replication has not been demonstrated. We have now used an antisense phosphorothioate oligonucleotide complementary to the intron-exon boundary of the UL36 and UL37 unspliced RNA to show that these gene products are required for HCMV DNA replication. Southern analysis showed that this oligonucleotide almost completely inhibits HCMV DNA replication when used at concentrations as low as 0.08 microM. The ability of this oligonucleotide to inhibit DNA replication was not the result of an inhibition of virus adsorption. Southern blots showed no impairment of viral adsorption or internalization in the presence of either specific or nonspecific phosphorothioate oligonucleotides. In addition, Northern (RNA) blots confirm that this antisense compound specifically reduced UL36 mRNA in treated cells to undetectable levels while the steady-state levels of immediate-early transcripts IE1 and IE2 were unaffected. These results demonstrate that the UL36 and UL37 gene products provide an essential function in initiation of HCMV DNA replication.

Amino Acid Sequence↗
PubMed1986

Mitomycin-induced chromatid breaks in HeLa cells: a consequence of incomplete DNA replication.

The formation of chromosome aberrations induced by alkylating agents such as mitomycin C has been shown to require the passage of the treated cell through S phase. However, the exact mechanisms by which mitomycin C-induced DNA lesions are translated into chromosome aberrations during S phase are not known. The purpose of these studies was to better understand the molecular basis of chromosome aberration formation after mitomycin C treatment. The morphology of metaphases of cells treated in G1 phase with mitomycin C resembled that of prematurely condensed chromosomes of S-phase cells. Consequently we postulated that chromosome aberrations resulted from cells reaching mitosis without completing DNA replication. This was tested by treating HeLa cells in G1 phase with mitomycin C and then analyzing these cells at mitosis for residual DNA damage and DNA content. Utilizing the DNA alkaline elution assay for DNA damage, we showed that HeLa cells progress through S phase into mitosis with intact DNA-DNA interstrand cross-links. These cross-links, originally induced into parental DNA, were associated equally with parental and newly replicated DNA at the time the cells reached mitosis. This suggests that recombinational events had taken place during the DNA replication process. Cells that were treated in G1 phase and allowed to proceed to mitosis in the presence of bromodeoxyuridine to density label newly replicated DNA were analyzed with cesium chloride density sedimentation. Unreplicated DNA was present in the mitotic cells of the treated populations but not in the untreated control cells. Further, flow cytometric measurements, made under hypotonic conditions in order to reduce chromatin condensation effects, demonstrated that the mitotic cells from the mitomycin C-treated populations contained 10-20% less DNA than untreated mitotic controls. These results indicate that chromosome breaks induced by mitomycin C are the result of cells reaching mitosis without having fully completed DNA replication.

Chromatids↗
PubMed1998

The acidic domain of pUL37x1 and gpUL37 plays a key role in transactivation of HCMV DNA replication gene promoter constructions.

Transient complementation of human cytomegalovirus (HCMV) oriLyt DNA replication in permissive human diploid cells expressing replication genes under native promoters requires its UL36-38 gene products. Two of the immediate early (IE) proteins encoded by this locus, pUL37x1 and, to a lesser extent, gpUL37, activated expression of HCMV early gene promoter constructions. The other IE protein encoded by the UL36-38 locus, pUL36, and the early product, pUL38, did not transactivate the HCMV early promoter constructions under similar conditions. The acidic domain, common to both pUL37x1 and gpUL37, is required for activation of HCMV early promoter constructions. Conversely, gpUL37 sequences downstream of amino acid 199 are not required for transactivation of viral early promoters. Taken together, these results suggest that the requirement for UL36-38 products for HCMV DNA replication results, at least in part, from the requirement of the transactivation of HCMV early DNA replication promoters by pUL37x1 and, to a lesser extent, by gpUL37 and that the acidic domain is critical for this activity.

Binding Sites↗
PubMed2003

The role of DNA recombination in herpes simplex virus DNA replication.

In many organisms the processes of DNA replication and recombination are closely linked. For instance, in bacterial and eukaryotic systems, replication forks can become stalled or damaged, in many cases leading to the formation of double stranded breaks. Replication restart is an essential mechanism in which the recombination and repair machinery can be used to continue replication after such a catastrophic event. DNA viruses of bacteria such as lambda and T4 also rely heavily on DNA recombination to replicate their genomes and both viruses encode specialized gene products which are required for recombination-dependent replication. In this review, we examine the linkage between replication and recombination in the eukaryotic pathogen, Herpes Simplex Virus Type 1 (HSV-1). The evidence that recombination plays an intrinsic role in HSV-1 DNA replication and the infection process will be reviewed. We have recently demonstrated that HSV-1 encodes two proteins which may be analogous to the lambda phage recombination system, Red(alpha) and beta. The HSV-1 alkaline nuclease, a 5' to 3' exonuclease, and ICP8, a single stranded DNA binding protein, can carry out strand annealing reactions similar to those carried out by the lambda Red system. In addition, evidence suggesting that host recombination proteins may also be important for HSV-1 replication will be reviewed. In summary, it is likely that HSV-1 infection will require both viral and cellular proteins which participate in various pathways of recombination and that recombination-dependent replication is essential for the efficient replication of viral genomes.

Bacteria↗