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At least 145 records · Page 8Linked to original sources
PubMed1999

Mechanism of DNA replication in eukaryotic cells: cellular host factors stimulating adenovirus DNA replication.

Replication of adenovirus (Ad) DNA depends on interactions between three viral and three cellular proteins. Human transcription factors NFI and Oct-1 recruit the Ad DNA polymerase to the origin of DNA replication as a complex with the Ad protein primer pTP. High affinity and specificity DNA binding to recognition sites in this origin by the transcription factors stimulate and stabilize pre-initiation complex formation to compensate for the low binding specificity of the pTP/pol complex. In this review, we discuss the properties of NFI and Oct-1 and the mechanism by which they enhance initiation of DNA replication. We propose a model that describes the dynamics of initiation and elongation as well as the assembly and disassembly of the pre-initiation complex.

Adenoviridae↗
PubMed1996

Identification of multiprotein complexes containing DNA replication factors by native immunoblotting of HeLa cell protein preparations with T-antigen-dependent SV40 DNA replication activity.

Increasing evidence has supported the concept that many of the enzymes and factors involved in the replication of mammalian DNA function together as a multiprotein complex. We have previously reported on the partial purification of a multiprotein form of DNA polymerase from human HeLa cells shown to be fully competent to support origin-specific large T-antigen-dependent simian virus 40 (SV40) DNA replication in vitro. In an attempt to more definitively identify the complex or complexes responsible for DNA replication in vitro, partially purified human HeLa cell protein preparations competent to replicate DNA in vitro were subjected to native polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose. The Native Western blots were probed with a panel of antibodies directed against proteins believed to be required for DNA replication in vitro. Apparent complexes of 620 kDa and 500 kDa were identified by monoclonal antibodies directed against DNA polymerase alpha and DNA polymerase delta, respectively. To detect epitopes possibly unexposed within the native multiprotein complexes, blots were also analyzed following denaturation in situ following treatment with detergent and reducing agent. The epitope or access to the epitope recognized by the monoclonal antibody against DNA polymerase alpha was destroyed by exposure of the blots to denaturing conditions. In contrast, an epitope present on a very large complex of approximately 1000 kDa was recognized by a monoclonal antibody against proliferating cell nuclear antigen only following treatment of the native immunoblots with denaturing agents. Identification of these complexes will allow their further purification, characterization, and elucidation of their role in the replication of DNA.

DNA Replication↗
PubMed1996

The expression of poly(ADP-ribose) polymerase during differentiation-linked DNA replication reveals that it is a component of the multiprotein DNA replication complex.

3T3-L1 preadipocytes have been shown to exhibit a transient increase in poly(ADP-ribose) polymerase (PARP) protein and activity, as well as an association of PARP with DNA polymerase alpha, within 12-24 h of exposure to inducers of differentiation, whereas 3T3-L1 cells expressing PARP antisense RNA showed no increase in PARP and are unable to complete the round of DNA replication required for differentiation into adipocytes. The role of PARP in differentiation-linked DNA replication has now been further clarified at both the cellular and enzymological levels. Flow cytometric analysis revealed that control 3T3-L1 cells progressed through one round of DNA replication prior to the onset of terminal differentiation, whereas cells expressing PARP antisense RNA were blocked at the G0/G1 phase of the cell cycle. Confocal microscope image analysis of control S phase cells demonstrated that PARP was localized within distinct intranuclear granular foci associated with DNA replication centers. On the basis of these results, purified replicative complexes from other cell types that had been characterized for their ability to catalyze viral DNA replication in vitro were analyzed for the presence of PARP. PARP exclusively copurified through a series of centrifugation and chromatography steps with core proteins of an 18-21S multiprotein replication complex (MRC) from human HeLa cells, as well as with the corresponding mouse MRC from FM3A cells. The MRC were shown to contain DNA polymerases alpha and delta, DNA primase, DNA helicase, DNA ligase, and topoisomerases I and II, as well as accessory proteins such as PCNA, RF-C, and RP-A. Finally, immunoblot analysis of MRCs from both cell types with monoclonal antibodies to poly (ADP-ribose) revealed the presence of approximately 15 poly(ADP-ribosyl)ated proteins, some of which were further confirmed to be DNA polymerase alpha, DNA topoisomerase I, and PCNA by immunoprecipitation experiments. These results suggest that PARP may play a regulatory role within the replicative apparatus as a molecular nick sensor controlling the progression of the replication fork or modulates component replicative enzymes or factors in the complex by directly associating with them or by catalyzing their poly(ADP-ribosyl)ation.

3T3 Cells↗
PubMed1991

Purification of DNA polymerase delta as an essential simian virus 40 DNA replication factor.

DNA replication from the SV40 origin can be reconstituted in vitro using purified SV40 large T antigen, cellular topoisomerases I and II, replication factor A (RF-A), proliferating cell nuclear antigen (PCNA), replication factor C (RF-C), and a phosphocellulose fraction (IIA) made from human cell extracts (S100). Fraction IIA contains all DNA polymerase activity required for replication in vitro in addition to other factors. A newly identified factor has been purified from fraction IIA. This factor is required for complete reconstitution of SV40 DNA replication and co-purifies with a PCNA-stimulated DNA polymerase activity. This DNA polymerase activity is sensitive to aphidicolin, but is not inhibited by butylanilinodeoxyadenosine triphosphate or by monoclonal antibodies which block synthesis by DNA polymerase alpha. The polymerase activity is synergistically stimulated by the combination of RF-A, PCNA, and RF-C in an ATP-dependent manner. Purified calf thymus polymerase delta can fully replace the purified factor in DNA replication assays. We conclude that this factor, required for reconstitution of SV40 DNA replication in vitro, corresponds to human DNA polymerase delta.

Cell Fractionation↗
PubMed1999

DRC1, DNA replication and checkpoint protein 1, functions with DPB11 to control DNA replication and the S-phase checkpoint in Saccharomyces cerevisiae.

In addition to DNA polymerase complexes, DNA replication requires the coordinate action of a series of proteins, including regulators Cdc28/Clb and Dbf4/Cdc7 kinases, Orcs, Mcms, Cdc6, Cdc45, and Dpb11. Of these, Dpb11, an essential BRCT repeat protein, has remained particularly enigmatic. The Schizosaccharomyces pombe homolog of DPB11, cut5, has been implicated in the DNA replication checkpoint as has the POL2 gene with which DPB11 genetically interacts. Here we describe a gene, DRC1, isolated as a dosage suppressor of dpb11-1. DRC1 is an essential cell cycle-regulated gene required for DNA replication. We show that both Dpb11 and Drc1 are required for the S-phase checkpoint, including the proper activation of the Rad53 kinase in response to DNA damage and replication blocks. Dpb11 is the second BRCT-repeat protein shown to control Rad53 function, possibly indicating a general function for this class of proteins. DRC1 and DPB11 show synthetic lethality and reciprocal dosage suppression. The Drc1 and Dpb11 proteins physically associate and function together to coordinate DNA replication and the cell cycle.

Amino Acid Sequence↗
PubMed1983

Analysis of simian virus 40 chromosome-T-antigen complexes: T-antigen is preferentially associated with early replicating DNA intermediates.

The fraction and DNA composition of simian virus 40 chromosomes that were complexed with large T-antigens (T-Ag) were determined at the peak of viral DNA replication. Simian virus 40 chromatin containing radiolabeled DNA was extracted by the hypotonic method of Su and DePamphilis (Proc. Natl. Acad. Sci. U.S.A. 73:3466-3470, 1976) and then fractionated by sucrose gradient sedimentation into replicating (90S) and mature (70S) chromosomes. Viral chromosomes containing T-Ag were isolated by immunoprecipitation with saturating amounts of either an anti-T-Ag monoclonal antibody or an anti-T-Ag hamster serum under conditions that specifically precipitated T-Ag protein from cytosol extracts. An average of 10% of the uniformly labeled DNA in the 90S pool and 7.5% in the 70S pool was specifically precipitated, demonstrating that under these conditions immunologically reactive T-Ag was tightly bound to only 8% of the total viral chromosomes. In contrast, simian virus 40 replicating intermediates (RI) represented only 1.2% of the viral DNA, but most of these molecules were associated with T-Ag. At the shortest pulse-labeling periods, an average of 72 +/- 18% of the radiolabeled DNA in 90S chromosomes could be immunoprecipitated, and this value rapidly decreased as the labeling period was increased. Electron microscopic analysis of the DNA before and after precipitation revealed that about 55% of the 90S chromosomal RI and 72% of the total RI from both pools were specifically bound to T-Ag. Comparison of the extent of replication with the fraction of RI precipitated revealed a strong selection for early replicating DNA intermediates. Essentially all of the RI in the 70S chromosomes were less than 30% replicated and were precipitated with anti-T-Ag monoclonal antibody or hamster antiserum. An average of 88% of the 90S chromosomal RI which were from 5 to 75% replicated were immunoprecipitated, but the proportion of RI associated with T-Ag rapidly decreased as replication proceeded beyond 70% completion. By the time sibling chromosomes had separated, only 3% of the newly replicated catenated dimers in the 90S pool (<1% of the dimers in both pools) were associated with T-Ag. Measurements of the fraction of radiolabeled DNA in each quarter of the genome confirmed that T-Ag was preferentially associated with newly initiated molecules in which the nascent DNA was nearest the origin of replication. These results are consistent with a specific requirement for the binding of T-Ag to viral chromosomes to initiate DNA replication, and they also demonstrate that T-Ag does not immediately dissociate from chromosomes once replication begins. The biphasic relationship between the fraction of T-Ag-containing RI and the extent of DNA replication suggests either that 1 or 2 molecules of T-Ag remain stably bound until replication is about 70% completed or that 4 to 6 molecules of T-Ag are randomly released from each RI at a uniform rate throughout replication.

Antibodies, Monoclonal↗
PubMed1992

Possible involvement of nuclear oncoproteins in regulation of DNA replication.

Polyomavirus DNA replication requires its enhancer which contains an AP-1 site. We have shown that protooncogenes, c-jun and c-fos, whose products form heterodimeric transcription activator, AP-1, strongly stimulate polyomavirus DNA replication through the AP-1 site. The mechanisms by which this enhancer stimulates replication and transcription are different. By replacing the enhancer with the oligonucleotides representing the binding site of a transcription factor of interest, any transcription factor with the known binding sequence can be characterized in this replication assay. When Rel protein was examined, we were able to reveal a new domain in v-Rel protein which can stimulate replication strongly. Whether this domain also coincides with transforming potential of v-Rel protein is currently under investigation.

Animals↗
PubMed1995

Comparison of autoradiography, liquid scintillation counting and immunoenzymatic staining of 5-bromo-2'-deoxyuridine for measurement of unscheduled DNA synthesis and replicative DNA synthesis in rat liver.

Different methods for evaluating unscheduled DNA synthesis (UDS) and replicative DNA synthesis (RDS) were studied in hepatocytes of F344 rats exposed in vivo to dimethylnitrosamine (DMN) or CCl4. Hepatocytes were isolated and incubated in Williams' medium E supplemented with either [3H]thymidine for autoradiography or 5-bromo-2'-deoxyuridine for immunoenzymatic staining. In the method of liquid scintillation counting, the cells were incubated with [3H]thymidine with or without hydroxyurea. The nuclear fraction was isolated and the incorporation of [3H]thymidine into nuclear DNA was determined by a liquid scintillation counter. DMN at doses of 0.625-5 mg/kg body weight induced UDS of 1.6-37.9 (0 dose; -6.9) net grains/nucleus measured by autoradiography and 337-1377 (0 dose; 177) dpm/microgram DNA in the presence of hydroxyurea measured by a liquid scintillation counter. CCl4 at doses of 50-400 mg/kg body weight induced RDS in 1.5-12.1% (0 dose; 0.12%) and 1.8-14.6% (0 dose; 0.16%) of cells with the methods of autoradiography and immunoenzymatic staining, respectively, and of 2991-24256 (0 dose; 324) dpm/microgram DNA in the absence of hydroxyurea with the method of liquid scintillation counting. Similar dose-dependent induction of UDS and RDS was observed with these methods. These results suggest that the methods of liquid scintillation counting and immunoenzymatic staining have almost the same sensitivity for measuring UDS and RDS as that of autoradiography.

Animals↗
PubMed1986

Differentiation of lens and neural cells in chicken embryos is accompanied by simultaneous decay of DNA replication machinery.

DNA polymerase alpha was detected in cells of developing chicken embryos by an immunofluorescent method using a monoclonal antibody specific for the high molecular weight polypeptide of chicken DNA polymerase alpha, and DNA polymerase beta was detected using a rabbit anti-chicken DNA polymerase beta antibody. In lens tissue of the 3- to 4-day chicken embryo, fluorescence with anti-DNA polymerase alpha antibody was detected in nuclei of lens epithelial cells but not in nuclei of lens fiber cells which had differentiated from epithelial cells. The localization of cells containing DNA polymerase alpha coincided with the distribution of cells capable of DNA replication as detected by [3H]thymidine autoradiography. Similar results were obtained during the differentiation of neural matrix cells to neuroblasts in the developing neural tube. In contrast to DNA polymerase alpha, DNA polymerase beta was detected in nuclei of both undifferentiated and differentiated cells of these tissues. Since the disappearance of DNA polymerase alpha was very rapid after the onset of differentiation, the DNA replication machinery in which DNA polymerase alpha plays a central role is thought to decay almost simultaneously with the onset of cellular differentiation in these tissues.

Animals↗
PubMed1982

The effect of aphidicolin on the rate of DNA replication and unscheduled DNA synthesis of Bloom syndrome and normal fibroblasts.

Ultraviolet radiation induced more unscheduled DNA synthesis (UDS) in ten Bloom syndrome (BS) fibroblast strains than in control cells, but this difference could be suppressed by aphidicolin treatment in at least nine BS strains. Aphidicolin, 1 and 5 micrograms/ml, were required to inhibit by 30% the UDS of BS and control cells respectively, but the DNA replication of BS cells did not prove abnormally sensitive to such an inhibitor. These findings are discussed in relation to current knowledge of the action of aphidicolin and hypotheses of the metabolic defect in BS.

Aphidicolin↗
PubMed1988

N-acetoxy-N-2-acetylaminofluorene-induced damage on SV40 DNA: inhibition of DNA replication and visualization of DNA lesions.

The chemical carcinogen, N-acetoxy-N-2-acetylaminofluorene (AAAF), which induces well characterized DNA lesions, strongly inhibits Simian virus 40 (SV40) DNA replication. By using SV40 mutants which were temperature-sensitive for replication initiation, we were able to synchronize SV40 DNA replication and therefore to introduce AAAF-induced lesions only on unreplicating SV40 molecules. One to two acetylaminofluorene (AAF)-adducts per SV40 genome inhibit more than 90% of normal semi-conservative DNA synthesis. SV40 replicative intermediates (RIs) from AAAF-treated infected cells, purified through neutral sucrose gradients and BND-cellulose column, possess a structure different from the usual Cairns molecules found in the untreated cultures. Both by neutral and alkaline sucrose gradients and by electron microscopy, the RIs isolated from treated cells appear as complex molecules with single-stranded portions and sometimes with a tailed structure. Moreover, the newly synthesized strands found in these molecules are equal in size to the average distance between AAF-adducts on the template strand, indicating that AAF-adducts represent a block for the SV40 DNA replication. By using specific anti Guo-AAF antibodies and electron microscopy, we show the presence of an AAF adduct at halted replication forks, i.e. showing a DNA replication block in a mammalian replicon for the first time. We therefore assume that AAF-adducts severely block the progression of the replication forks by inhibiting, at least, the in vivo polymerization of the leading strand.

2-Acetylaminofluorene↗
PubMed1999

CDC45 and DPB11 are required for processive DNA replication and resistance to DNA topoisomerase I-mediated DNA damage.

The antitumor agent camptothecin targets DNA topoisomerase I by reversibly stabilizing a covalent enzyme-DNA intermediate. The subsequent collision of DNA replication forks with these drug-enzyme-DNA complexes produces the cytotoxic DNA lesions that signal cell cycle arrest and ultimately lead to cell death. Despite intense investigation, the character of the lesions produced and the repair processes that resolve the damage remain poorly defined. A yeast genetic screen was implemented to isolate conditional mutants with enhanced sensitivity to DNA topoisomerase I-mediated DNA damage. Cells exhibiting temperature-sensitive growth in the presence of the DNA topoisomerase I mutant, Top1T722Ap, were selected. Substitution of Ala for Thr722 increases the stability of the covalent Top1T722Ap-DNA intermediate, mimicking the cytotoxic action of camptothecin. Two mutants isolated, cdc45-10 and dpb11-10, exhibited specific defects in DNA replication and a synthetic lethal phenotype in the absence of DNA damaging agents. The accumulation of Okazaki fragments under nonpermissive conditions suggests a common function in promoting processive DNA replication through polymerase switching. These results provide a mechanistic basis for understanding the cellular processes involved in the resolution of DNA damage induced by camptothecin and DNA topoisomerase I.

Camptothecin↗
PubMed1990

Inhibition of replicative DNA synthesis and induction of DNA repair in human fibroblasts by the intercalating drugs proflavine and 9-aminoacridine.

The induction of unscheduled DNA synthesis (UDS) and the alteration of semiconservative DNA replication by the structurally related intercalating agents proflavine and 9-aminoacridine were studied in MRC-5 human fibroblasts in culture. Autoradiographic determinations of both parameters were carried out simultaneously in the same culture specimens. Proflavine affected DNA synthesis, but did not elicit any UDS. 9-Aminoacridine inhibited DNA synthesis only at the highest concentration and caused UDS to a low but significant extent. These results suggest that the ability to induce UDS is not a general property of the intercalating agents and that the alterations of the DNA structure, typical of the "pure" intercalative process, are not handled by pathways involving unscheduled synthesis.

Aminacrine↗
PubMed1990

DNA replication studies with coliphage 186. III. A single phage gene is required for phage 186 replication.

We have shown that the BglII to BamHI (79.6% to 95.8%) region of the coliphage 186 chromosome can direct 186-specific replication. DNA sequencing of the region revealed five presumptive genes, CP80, CP81, CP83, CP84 and CP87. Surprisingly, alleles of the previously defined replication gene, A, were localized in both CP84 and CP87. We have successfully constructed a 186 minichromosome using the single gene CP87, and determined that CP84 was not concerned with replication, neither of a minichromosome nor of the phage. Rather, the replication defect seen with amber mutants of CP84 reflects a polarity effect on the downstream expression of CP87. We have concluded that CP87 is the only phage gene necessary for 186 replication, and have called it gene A.

Amino Acid Sequence↗