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At least 1,711 records · Page 95Linked to original sources
PubMed1984

Control of DNA replication and cell proliferation in eukaryotes by aphidicolin.

The mycotoxin aphidicolin specifically inhibits nuclear DNA synthesis in eukaryotic cells by intereacting with the replicative DNA polymerase alpha. The drug does not bind directly to DNA nor does it interfere with RNA, protein and nucleic acid precursor synthesis. Aphidicolin offers a new tool for analyzing the mechanism of DNA replication and repair in eukaryotes and for studying the role of eukaryotic DNA polymerases. The drug might also be valuable therapeutically for controlling excessive cell proliferation without adverse effects upon non-multiplying cells. We describe here how to synchronize DNA synthesis in human cells by a double block with aphidicolin and the properties of two human cell lines resistant to the drug.

Animals↗
PubMed2003

The integral membrane protein p16.7 organizes in vivo phi29 DNA replication through interaction with both the terminal protein and ssDNA.

Remarkably little is known about the in vivo organization of membrane-associated prokaryotic DNA replication or the proteins involved. We have studied this fundamental process using the Bacillus subtilis phage phi29 as a model system. Previously, we demonstrated that the phi29-encoded dimeric integral membrane protein p16.7 binds to ssDNA and is involved in the organization of membrane-associated phi29 DNA replication. Here we demonstrate that p16.7 forms multimers, both in vitro and in vivo, and interacts with the phi29 terminal protein. In addition, we show that in vitro multimerization is enhanced in the presence of ssDNA and that the C-terminal region of p16.7 is required for multimerization but not for ssDNA binding or interaction with the terminal protein. Moreover, we provide evidence that the ability of p16.7 to form multimers is crucial for its ssDNA-binding mode. These and previous results indicate that p16.7 encompasses four distinct modules. An integrated model of the structural and functional domains of p16.7 in relation to the organization of in vivo phi29 DNA replication is presented.

Bacillus Phages↗
PubMed1984

Mechanism of control of DNA replication and incompatibility in ColE1-type plasmids--a review.

The related phenomena of ColE1 DNA replication control and plasmid incompatibility are analysed in detail. Replication is negatively regulated in different ways by two gene products, RNA I and gene rop-coded polypeptide, while plasmid incompatibility is specified only by RNA I. Thus, copy-number mutants in the RNA I region usually have altered incompatibility and vice versa. RNA I is transcribed from the opposite strand to RNA II; the processed RNA II transcript serves as the primer for DNA replication. The base-pairing interaction between RNA I and RNA II controls DNA synthesis and incompatibility by preventing primer formation. A model is proposed to explain the phenotype of RNA I mutants in terms of the changes they cause in the secondary structure of RNA I or RNA II.

Bacteriocin Plasmids↗
PubMed1989

Yeast replication factor-A functions in the unwinding of the SV40 origin of DNA replication.

Cell-free replication systems for simian virus 40 (SV40) DNA are taken to be a model for the replication of eukaryotic chromosomes, because only one viral protein is required to supplement the replication proteins provided by a human cell extract. To prove that these cellular proteins function in chromosomal DNA replication we have begun to identify homologous proteins in an organism that can be genetically manipulated. Here we report the identification of yeast replication factor-A (yRF-A) from Saccharomyces cerevisiae and show that it is functionally and structurally related to a human protein that is required for the initiation and elongation of SV40 DNA replication. Yeast RF-A, a multi-subunit phosphoprotein, is similar to the human protein in its chromatographic behaviour, subunit structure and DNA-binding activity. The yeast protein will fully substitute for the human protein in an early stage of the initiation of SV40 DNA replication. Substitution of yRF-A in the complete SV40 replication system, however, results in reduced DNA replication, presumably due to a requirement for species-specific interactions between yeast RF-A and the DNA polymerase complex.

Binding, Competitive↗
PubMed1982

Formation of a new DNA replication intermediate in novobiocin-treated human melanoma cells.

The effect of novobiocin on the formation of DNA replication intermediates in human melanoma cells was investigated. This drug reduces DNA synthesis in all probability by interfering with the DNA gyrases (enzymes that are involved in the supercoiling of the DNA). The single-stranded DNA replication intermediates were released from the parental DNA by lysing the cells in dilute alkali and were then fractionated by agarose gel electrophoresis. In the untreated cells one can detect replication intermediates ranging in size from Okazaki-fragments up to 10 kb. However, in novobiocin-treated cells, apart from the already known intermediates, formation of a new replication intermediate with the size of about 20 kb was also detected. The 20-kb DNA shows a similar discrete appearance in the agarose gel as the 10-kb DNA intermediate.

Cells, Cultured↗
PubMed1977

The nucleotide sequence surrounding the origin of DNA replication of Col E1.

The DNA of Col E1 replicates from a unique origin located at a distance of 17-19% of the genome length from the single Eco RI clevage site. The nucleotide sequence about this site has been determined by a combination of RNA and DNA sequencing techniques. The principal features of the sequence are two palindromes, one of which resembles a palindrome located in the intercistronic region of 0X174. The sequence also contains stretches of purine and pyrimidine clusters of the following compositions: pAT5G, pC2T5G, pGT5G. The origin sequence demonstrates that initiation of DNA replication takes place in an intercistronic region of Col E1DNA, although the possibility that this region makes small polypeptides 30-40 residues long cannot be strictly eliminated at this time.

Bacteriocin Plasmids↗
PubMed2003

Reactivation of lytic replication from B cells latently infected with Epstein-Barr virus occurs with high S-phase cyclin-dependent kinase activity while inhibiting cellular DNA replication.

Productive infection and replication of herpesviruses usually occurs in growth-arrested cells, but there has been no direct evidence in the case of Epstein-Barr virus (EBV), since an efficient lytic replication system without external stimuli does not exist for the virus. Expression of the EBV lytic-switch transactivator BZLF1 protein in EBV-negative epithelial tumor cell lines, however, is known to arrest the cell cycle in G(0)/G(1) by induction of the tumor suppressor protein p53 and the cyclin-dependent kinase (CDK) inhibitors p21(WAF-1/CIP-1) and p27(KIP-1), followed by the accumulation of a hypophosphorylated form of the Rb protein. In order to determine the effect of the onset of lytic viral replication on cellular events in latently EBV-infected B LCLs, a tightly controlled induction system of the EBV lytic-replication program by inducible BZLF1 protein expression was established in B95-8 cells. The induction of lytic replication completely arrested cell cycle progression and cellular DNA replication. Surprisingly, the levels of p53, p21(WAF-1/CIP-1), and p27(KIP-1) were constant before and after induction of the lytic program, indicating that the cell cycle arrest induced by the lytic program is not mediated through p53 and the CDK inhibitors. Furthermore, although cellular DNA replication was blocked, elevation of cyclin E/A expression and accumulation of hyperphosphorylated forms of Rb protein were observed, a post-G(1)/S phase characteristic of cells. Thus, while the EBV lytic program promoted specific cell cycle-associated activities involved in the progression from G(1) to S phase, it inhibited cellular DNA synthesis. Such cellular conditions appear to especially favor viral lytic replication.

B-Lymphocytes↗
PubMed1992

Nuclear factor I enhances adenovirus DNA replication by increasing the stability of a preinitiation complex.

Nuclear factor I (NFI) or its isolated DNA-binding domain (NFI-BD) enhances initiation of adenovirus DNA replication up to 50-fold at low concentrations of the precursor terminal protein-DNA polymerase (pTP-pol) complex. Both in solution and when bound to DNA, NFI-BD can form a complex with pTP-pol. To investigate the mechanism of enhancement by NFI, we determined the stability of a functional preinitiation complex formed in vitro between pTP-pol and the origin. Challenge experiments with a distinguishable template containing an identical origin revealed that in the absence of NFI, this preinitiation complex was very sensitive to competition for pTP-pol. Addition of NFI-BD increased the half-life of the complex at least 10-fold and led to the formation of a template-committed preinitiation complex. In agreement with this, binding of pTP-pol to origin DNA in band-shift assays was enhanced by NFI. By DNase I footprinting we show that the specificity of binding as well as induction of structural changes in origin DNA by pTP-pol are increased by NFI. These results indicate that NFI, by binding and positioning pTP-pol, stabilizes the complex between pTP-pol and the core origin, and thus enhances initiation of DNA replication.

Adenoviruses, Human↗
PubMed1982

Initiation of bacteriophage lambda DNA replication in vitro with purified lambda replication proteins.

We have developed a soluble enzyme system that replicates exogenously added plasmid DNA (lambda dv) bearing the replication origin of the bacteriophage lambda chromosome. The system contains pure phage lambda O and P replication proteins and a partially purified mixture of Escherichia coli replication proteins [the enzyme system of Fuller, R.S., Kaguni, J.M. & Kornberg, A. (1981) Proc. Natl. Acad. Sci. USA 78, 7370-7374). The features of lambda dv replication in this system closely resemble the known characteristics of phage lambda DNA replication in vivo. The system (i) depends completely on exogenously supplied DNA, (ii) specifically replicates supercoiled plasmid DNA that contains a lambda replication origin, (iii) depends on both the lambda O protein and the lambda P protein, (iv) depends on RNA polymerase, (v) depends on host replication proteins (e.g., primase, dnaB protein, and several others that function in the priming of DNA synthesis in E. coli) as judged by antibody inhibitions, and (vi) replicates as much as 32% of added lambda dv plasmid DNA through a single complete round to generate catenated daughter molecules. Furthermore, replication of lambda dv DNA in vitro requires DNA gyrase and an ATP-regenerating system. It is notable that addition of lambda O and P proteins to the mixture of E. coli replication proteins inhibits replication of plasmids bearing the origin of the E. coli chromosome. Exploitation of this enzyme system should allow a detailed investigation of the biochemical mechanisms involved in bacteriophage lambda DNA replication and its regulation.

Bacteriophage lambda↗
PubMed1997

The role of inhibitory phosphorylation of CDC2 following DNA replication block and radiation-induced damage in human cells.

It has been suggested that the survival response of p53 defective tumor cells to agents that inhibit DNA replication or damage DNA may be largely dependent on cell cycle checkpoints that regulate the onset of mitosis. In human cells, the mitosis-inducing kinase CDC2/cyclin B is inhibited by phosphorylation of threonine-14 and tyrosine-15, but the roles of these phosphorylations in enforcing checkpoints is not known. We have investigated the situation in a human cervical carcinoma cell line (HeLa cells) and found that low level expression of a mutant nonphosphorylatable form of CDC2 abrogates regulation of the endogenous CDC2/cyclin B. Disruption of this pathway is toxic and renders cells highly sensitive to killing by DNA damage or by inhibition of DNA replication. These findings establish the importance of inhibitory phosphorylation of CDC2 in the survival mechanism used by human cells when exposed to some of the most common forms of anticancer therapy.

CDC2 Protein Kinase↗
PubMed2003

Distinct roles of the Adenovirus E4 ORF3 protein in viral DNA replication and inhibition of genome concatenation.

Adenovirus early proteins E4 ORF3 and E4 ORF6 have complementary functions during viral infection. Both proteins facilitate efficient viral DNA replication, late protein expression, and prevention of concatenation of viral genomes. Additionally, E4 ORF6 is involved in the shutoff of the host cell protein synthesis through its interaction with the E1B 55K protein. This complex also leads to the degradation of p53. A unique function of E4 ORF3 is the reorganization of nuclear structures known as PML oncogenic domains (PODs). The function of these domains is unclear, but PODs have been implicated in a number of important cellular processes, including transcriptional regulation, apoptosis, transformation, and response to interferon. The goal of this study was to determine the functional significance of the reorganization of PODs by E4 ORF3. Point mutations were made in the E4 ORF3 gene. These mutants were recombined into a virus lacking E4 ORF6 and expressed under the control of the natural virus E4 promoter. The panel of mutant viruses was used to investigate the role of E4 ORF3 during the course of the viral infection program. One of the mutant viruses exhibited aberrant reorganization of PODs and had a severe defect in viral DNA replication, thus leading to a dramatic decrease in virus production. A number of mutants accumulated viral DNA and infectious virus particles to wild-type levels but showed significant viral genome concatenation. These data show that E4 ORF3 is a multifunctional protein and that a specific rearrangement of nuclear PML domains is coupled to efficient viral DNA replication. This function is distinct from the role of E4 ORF3 in the regulation of virus genome concatenation via inhibition of cellular double-strand break repair.

Adenovirus E4 Proteins↗
PubMed1990

Extrachromosomal recombination in vaccinia-infected cells requires a functional DNA polymerase participating at a level other than DNA replication.

Homologous recombination was measured in vaccinia-infected cells cotransfected with two plasmid recombination substrates. One plasmid contains a vaccinia protein lacZ coding region bearing a 1.1 kb 3' terminal deletion while the other plasmid contains a non-promoted lacZ coding region bearing a 1.1 kb 5' terminal deletion. Homologous recombination occurring between the 825 bp of lacZ common to both plasmids regenerates a functional lacZ gene from which B-galactosidase expression was measured. The entire 3 kb lacZ gene was used as a positive control. A panel of thermosensitive mutants was screened in cells either transfected with the positive control plasmid or cotransfected with the recombination substrates. A DNA - mutant, ts42, known to map to the viral DNA polymerase gene was found to be defective in recombination. Significantly, other DNA - mutants, ts17 or ts25, or other DNA polymerase mutants did not exhibit a defect in recombination similar to ts42. Inhibitors of viral DNA synthesis did not uniformly affect recombination. Cytosine arabinoside and aphidicolin inhibited B-galactosidase expression from the recombination substrates but not from the positive control plasmid, whereas hydroxyurea enhanced expression from both. Marker rescue with the cloned wildtype DNA polymerase gene repaired the defect in ts42. Southern and western analyses demonstrated that B-galactosidase activity was consistent with a recombined lacZ gene and unit size 116 kDa protein. Measurement of plasmid and viral DNA replication in cells infected with the different DNA - mutants indicated that recombination was independent of plasmid and viral DNA replication. Together these results suggest that the vaccinia DNA polymerase participates in homologous recombination at a level other than that of DNA replication.

Animals↗
PubMed1993

Nucleotide sequence of the primer RNA for DNA replication of filamentous bacteriophages.

We determined the nucleotide sequence of RNA synthesized in vitro by Escherichia coli RNA polymerase at the complementary-strand replication origin on the single-stranded viral DNA of bacteriophages f1 and IKe (ori-RNA) by using chain-terminating ribonucleoside triphosphate analogs. The results indicated that the start site of f1 ori-RNA synthesis is 20 nucleotides downstream from the site previously reported (K. Geider, E. Beck, and H. Schaller, Proc. Natl. Acad. Sci. USA 75:645-649, 1978) and that the RNA sequence [(5')pppAGGGCGAUGGCCCACUACGU-OH(3')] is complementary to the f1 DNA sequence from nucleotides 5736 to 5717, with minor heterogeneity at the 3' end. IKe ori-RNA had a sequence identical to that of f1 ori-RNA, except for a single base substitution, and IKe RNA was complementary to a region of IKe DNA (from nucleotides 6441 to 6422) that was homologous to the f1 sequence. Phenotypes and ori-RNA sequences in the relevant region of the genome of f1 deletion mutants were consistent with the presently determined sequence of ori-RNA. A possibility that ori-RNA synthesis is initiated by a mechanism similar to that for general transcription is suggested as a result of the new assignment of the ori-RNA start site. The double-origin plasmid assay of minus-strand origin activity, a sensitive in vivo method for detecting cis-acting elements for the initiation of DNA replication on a single-stranded DNA template, is described.

Base Sequence↗
PubMed1982

Studies on differentiation without cleavage in Chaetopterus. Requirement for a definite number of DNA replication cycles shown by aphidicolin pulses.

Aphidicolin, a powerful inhibitor of nuclear DNA replication, has been used to establish the level of polyploidisation required for the realization of the late morphogenetic events (segregation, pseudogastrulation and ciliation) of differentiation without cleavage in Chaetopterus-activated eggs. A parallel has been drawn between cytophotometric estimations of the DNA content and cytoplasmic differentiation in eggs treated with aphidicolin under different experimental conditions: either pulses with aphidicolin were followed by culture in sea water or the eggs were treated with aphidicolin after development had taken place in sea water for various lengths of time. The results suggest that a 'quantal' monasterial cycle might take place 3 h after activation, corresponding presumably to the fourth or fifth replication cycle. Moreover, early DNA replication seems to be more important for morphogenesis than late DNA replication.

Animals↗
PubMed2002

Knotting dynamics during DNA replication.

The topology of plasmid DNA changes continuously as replication progresses. But the dynamics of the process remains to be fully understood. Knotted bubbles form when topo IV knots the daughter duplexes behind the fork in response to their degree of intertwining. Here, we show that knotted bubbles can form during unimpaired DNA replication, but they become more evident in partially replicated intermediates containing a stalled fork. To learn more about the dynamics of knot formation as replication advances, we used two-dimensional agarose gel electrophoresis to identify knotted bubbles in partially replicated molecules in which the replication fork stalled at different stages of the process. The number and complexity of knotted bubbles rose as a function of bubble size, suggesting that knotting is affected by both precatenane density and bubble size.

DNA Replication↗
PubMed1995

Eukaryotic DNA replication.

Recent discoveries suggest that the initiation of eukaryotic DNA replication involves at least two steps--one occurring near the completion of mitosis and the other at the onset of S phase--that bring about the ordered assembly of initiator proteins at the origin. The identification and characterization of components involved in promoting or antagonizing each of these steps has provided a preliminary understanding of how replication initiation is regulated so precisely during the cell cycle.

Animals↗
PubMed2005

Glucose inhibits meiotic DNA replication through SCFGrr1p-dependent destruction of Ime2p kinase.

In the budding yeast Saccharomyces cerevisiae, the cell division cycle and sporulation are mutually exclusive cell fates; glucose, which stimulates the cell division cycle, is a potent inhibitor of sporulation. Addition of moderate concentrations of glucose (0.5%) to sporulation medium did not inhibit transcription of two key activators of sporulation, IME1 and IME2, but did increase levels of Sic1p, a cyclin-dependent kinase inhibitor, resulting in a block to meiotic DNA replication. The effects of glucose on Sic1p levels and DNA replication required Grr1p, a component of the SCF(Grr1p) ubiquitin ligase. Sic1p is negatively regulated by Ime2p kinase, and several observations indicate that glucose inhibits meiotic DNA replication through SCF(Grr1p)-mediated destruction of this kinase. First, Ime2p was destabilized in the presence of glucose, and this turnover required Grr1p, a second component of SCF(Grr1p), Cdc53p, and an SCF(Grr1p)-associated E2 enzyme, Cdc34p. Second, Ime2p-ubiquitin conjugates were detected under conditions of rapid Ime2p turnover, and conjugation of Ime2p to ubiquitin required GRR1. Third, a mutant form of Ime2p (Ime2(DeltaPEST)), in which a putative Grr1p-interacting sequence was deleted, was more stable than wild-type Ime2p. Finally, expression of the IME2(DeltaPEST) allele bypassed the block to meiotic DNA replication caused by 0.5% glucose. In addition, Grr1p is required for later events in sporulation independently of its role in Ime2p turnover.

Alleles↗
PubMed1998

The role of the ran GTPase in nuclear assembly and DNA replication: characterisation of the effects of Ran mutants.

The Ran GTPase plays a critical role in nucleocytoplasmic transport and has been implicated in the maintenance of nuclear structure and cell cycle control. Here, we have investigated its role in nuclear assembly and DNA replication using recombinant wild-type and mutant Ran proteins added to a cell-free system of Xenopus egg extracts. RanQ69L and RanT24N prevent lamina assembly, PCNA accumulation and DNA replication. These effects may be due to the disruption of nucleocytoplasmic transport, since both mutants inhibit nuclear import of a protein carrying a nuclear localisation signal (NLS). RanQ69L, which is deficient in GTPase activity, sequesters importins in stable complexes that are unable to support the docking of NLS-proteins at the nuclear pore complex (NPC). RanT24N, in contrast to wild-type Ran-GDP, interacts only weakly with importin alpha and nucleoporins, and not at all with the import factor p10, consistent with its poor activity in nuclear import. However, RanT24N does interact stably with importin beta, Ran binding protein 1 and RCC1, an exchange factor for Ran. We show that Ran-GDP is essential for proper nuclear assembly and DNA replication, the requirement being primarily before the initiation of DNA replication. Ran-GDP therefore mediates the active transport of necessary factors or otherwise controls the onset of S-phase in this system.

Animals↗