Effect of aphidicolin on the elongation step of adenovirus DNA replication in vitro

Adenovirus DNA synthesis carried out in vitro was inhibited by the aphidicolin. However, 30% of the DNA synthesis was resistant to aphidicolin even at a concentration of 200 micrograms/ml. When the distribution patterns of the radioactivity of the products synthesized in the presence of 50 micrograms/ml of the drug was examined after HindIII digestion of the product DNA, the radioactivity appeared preferentially in the fragments mapping nearest to the ends of the molecule. Pulse-chase experiment showed that the terminal fragments were synthesized with or without aphidicolin but that in the presence of aphidicolin the rate of elongation rapidly slowed down beyond this region, suggesting that a DNA polymerase sensitive to aphidicolin may participate in the synthesis of the internal region of adenovirus DNA.     

The effect of aphidicolin on adenovirus DNA synthesis.

Aphidicolin inhibits adenovirus DNA replication in HeLa cells and in a cell-free, infected, nuclear extract in which viral DNA is elongated. The compound inhibits alpha DNA polymerase, extensively purified from HeLa cells, but has little or no effect on the beta or gamma DNA polymerases similarly purified. Aphidicolin does not affect thymidine uptake by cells nor does synthesis as it also inhibits DNA replication in uninfected cells. The inhibition by aphidicolin is reversible if the drug is removed within 18 hrs after addition to HeLa or Chinese Hamster Ovary cells but the cells are irreversibly affected if the drug remains for 48 hours.     

The adenovirus priming protein pTP contributes to the kinetics of initiation of DNA replication

Adenovirus (Ad) precursor terminal protein (pTP) in a complex with Ad DNA polymerase (pol) serves as a primer for Ad DNA replication. During initiation, pol covalently couples the first dCTP with Ser-580 of pTP. By using an in vitro reconstituted replication system comprised of purified proteins, we demonstrate that the conserved Asp-578 and Asp-582 residues of pTP, located close to Ser-580, are important for the initiation activity of the pTP/pol complex. In particular, the negative charge of Asp-578 is essential for this process. The introduced pTP mutations do not alter the binding capacity to DNA or polymerase, suggesting that the priming mechanism is affected. The Asp-578 or Asp-582 mutations increase the K_m for dCTP incorporation, and higher dCTP concentrations or Mn²⁺ replacing Mg²⁺ partially relieve the initiation defect. Moreover, the k_cat/K_m values are reduced as a consequence of the pTP mutations. These observations demonstrate that pTP influences the catalytic activity of pol in initiation. Since both Asp residues are situated close to the pol active site during initiation, they may contribute to correct positioning of the OH group in Ser-580. Our results indicate that specific amino acids of the protein primer influence the ability of Ad5 DNA polymerase to initiate DNA replication.     

Differential effect of aphidicolin on adenovirus DNA synthesis and cellular DNA synthesis.

There is strong evidence for a participation of DNA polymerase gamma in the replication of adenovirus (Ad) DNA. To study a possible additional role of DNA polymerase alpha we measured the effect of aphidicolin on viral DNA replication. In intact cells, aphidicolin inhibits Ad DNA synthesis weakly. The drug concentration required for 50% inhibition of Ad DNA replication was 300-400 fold higher than for a similar effect on cellular DNA synthesis. Such a differential inhibition was also observed in AGMK cells doubly infected with SV40 and the simian adenovirus SA7. No evidence was found for modification of aphidicolin in infected cells or for a change in aphidicolin sensitivity of DNA polymerase alpha after infection. The extent of inhibition of purified DNA polymerase alpha was dependent upon the dCTP concentration. The same situation was observed when DNA synthesis was studied in isolated nuclei from uninfected cells. However, in nuclei from Ad infected cells no effect of dCTP on aphidicolin sensitivity was found. These results were taken as evidence that DNA polymerase alpha does not participate in the replication of adenovirus DNA.     

DNA sequences required for the initiation of adenovirus type 4 DNA replication in vitro

In-vivo studies have demonstrated that adenovirus type 2 and adenovirus type 4 have different DNA sequence requirements for the initiation of DNA replication. To investigate the basis of these differences an in-vitro system has been developed which will faithfully initiate adenovirus type 4 DNA replication. A plasmid containing 140 base-pairs of the right terminus of adenovirus type 4 supported initiation of DNA replication in vitro, provided that the plasmid was linearized in such a way as to locate the viral terminal sequences at the molecular ends of the DNA. Initiation by adenovirus type 4-infected cell extracts was also supported by a plasmid containing the complete adenovirus type 2 inverted terminal repeat (ITR). Deletion analysis of both adenovirus types 2 and 4 ITRs revealed that only the terminal 18 base-pairs of the genomes (perfectly conserved between the 2 viruses) were required for initiation in vitro. Thus, initiation was not enhanced by the presence of either the NFI site, the NFIII site or both sites together. Fractionation of a HeLa cell nuclear extract, by ion-exchange chromatography, identified a nuclear factor that stimulated the initiation reaction four- to fivefold. The stimulatory factor did not correspond to either of the cellular proteins NFI or NFIII which stimulate adenovirus type 2 DNA replication in vitro. Initiation in vitro was also supported by single-stranded DNA templates, albeit at a lower efficiency. Studies with synthetic oligonucleotides indicated a surprising specificity for initiation: whereas the strand used as template during initiation in vivo was active as a template for initiation in vitro, the complementary strand was inactive.     

Characterization of an adenovirus early protein required for viral DNA replication: A single strand specific DNA binding protein

1. The human adenoviruses types 2, 5 and 12 code for the production of a single strand specific DNA binding protein. The molecular weights of these proteins were 72,000 for types 2 and 5 and 60,000 for type 12. In all three cases proteolytic breakdown fragments of these binding proteins (48,000 MW) were also observed. 2. Analysis of the methionine containing tryptic peptides of these proteins indicate that the types 2 and 5 proteins are similar and clearly distinguishable from the type 12 protein. The peptide maps of these three viral proteins are clearly different from a similar protein found in mock infected cells. 3. Temperature sensitive mutants of type 5 (H5ts125) and type 12(H12tsA275) adenoviruses fail to produce these proteins at the nonpermissive temperature. H5ts125 infected cells grown at the permissive temperature produce a 72,000 MW protein that is thermolabile, for continued binding to DNA, when compared to type 5 wild type adenovirus 72,000 MW protein. An analysis of the phenotype of this adenovirus mutant indicates that it codes for a viral function at early times after infection that is required for viral DNA replication. 4. The in vitro translation of adenovirus specific m-RNA results in the synthesis of a small amount of a 72,000 MW protein that binds to single stranded DNA just like the authentic adenovirus DNA binding proteins produced in infected cells. 5. Adenovirus anti-Tumor antigen (T) anti-serum from hamsters carrying independently derived adenovirus tumors, have been tested for the presence of antibody to purified DNA binding proteins. One antiserum is positive for these antibodies while the other is negative. These results indicate that some, but not all, adenovirus tumors contain large enough levels of the DNA binding proteins to elicit an antibody response. 6. The type 5 adenovirus temperature sensitive mutant, H5ts125, that codes for a thermolabile DNA binding protein, was complemented or suppressed at the nonpermissive temperature, for the replication of adenovirus DNA, by SV40. SV40tsA temperature sensitive mutants, defective in SV40 DNA replication, do not suppress or complement H5ts125 at the nonpermissive temperature.     

DNA binding properties of the adenovirus DNA replication priming protein pTP

The precursor terminal protein pTP is the primer for the initiation of adenovirus (Ad) DNA replication and forms a heterodimer with Ad DNA polymerase (pol). Pol can couple dCTP to pTP directed by the fourth nucleotide of the viral genome template strand in the absence of other replication proteins, which suggests that pTP/pol binding destabilizes the origin or stabilizes an unwound state. We analyzed the contribution of pTP to pTP/pol origin binding using various DNA oligonucleotides. We show that two pTP molecules bind cooperatively to short DNA duplexes, while longer DNA fragments are bound by single pTP molecules as well. Cooperative binding to short duplexes is DNA sequence independent and most likely mediated by protein/protein contacts. Furthermore, we observed that pTP binds single-stranded (ss)DNA with a minimal length of approximately 35 nt and that random ssDNA competed 25-fold more efficiently than random duplex DNA for origin binding by pTP. Remarkably, short DNA fragments with two opposing single strands supported monomeric pTP binding. pTP did not stimulate, but inhibited strand displacement by the Ad DNA binding and unwinding protein DBP. These observations suggest a mechanism in which the ssDNA affinity of pTP stabilizes Ad pol on partially unwound origin DNA.     

Molecular architecture of adenovirus DNA polymerase and location of the protein primer

Adenovirus (Ad) DNA polymerase (pol) belongs to the distinct subclass of the polalpha family of DNA pols that employs the precursor terminal protein (pTP) as primer. Ad pol forms a stable heterodimer with this primer, and together, they bind specifically to the core origin in order to start replication. After initiation of Ad replication, the resulting pTP-trinucleotide intermediate jumps back and pTP starts to dissociate. Compared to free Ad pol, the pTP-pol complex shows reduced polymerase and exonuclease activities, but the reason for this is not understood. Furthermore, the interaction domains between these proteins have not been defined and the contribution of each protein to origin binding is unclear. To address these questions, we used oligonucleotides with a translocation block and show here that pTP binds at the entrance of the primer binding groove of Ad pol, thereby explaining the decreased synthetic activities of the pTP-pol complex and providing insight into how pTP primes Ad replication. Employing an exonuclease-deficient mutant polymerase, we further show that the polymerase and exonuclease active sites of Ad pol are spatially distinct and that the exonuclease activity of Ad pol is located at the N-terminal part of the protein. In addition, by probing the distances between both active sites and the surface of Ad pol, we show that Ad pol binds a DNA region of 14 to 15 nucleotides. Based on these results, a model for binding of the pTP-pol complex at the origin of replication is proposed.     

Simian virus 40 DNA replication in vitro: specificity of initiation and evidence for bidirectional replication.

We recently described a soluble cell-free system derived from monkey cells that is capable of replicating exogenous plasmid DNA molecules containing the simian virus 40 (SV40) origin of replication (J.J. Li, and T.J. Kelly, Proc. Natl. Acad. Sci. U.S.A. 81:6973-6977, 1984). Replication in the system is completely dependent upon the addition of the SV40 large T antigen. In this report we describe additional properties of the in vitro replication reaction. Extracts prepared from cells of several nonsimian species were tested for the ability to support origin-dependent replication in the presence of T antigen. The activities of extracts derived from human cell lines HeLa and 293 were approximately the same as those of monkey cell extracts. Chinese hamster ovary cell extracts also supported SV40 DNA replication in vitro, but the extent of replication was approximately 1% of that observed with human or monkey cell extracts. No replication activity was detectable in extracts derived from BALB/3T3 mouse cells. The ability of these extracts to support replication in vitro closely parallels the ability of the same cells to support replication in vivo. We also examined the ability of various DNA molecules containing sequences homologous to the SV40 origin to serve as templates in the cell-free system. Plasmids containing the origins of human papovaviruses BKV and JCV replicated with an efficiency 10 to 20% of that of plasmids containing the SV40 origin. Plasmids containing Alu repeat sequences (BLUR8) did not support detectable DNA replication in vitro. Circular DNA molecules were found to be the best templates for DNA replication in the cell-free system; however, linear DNA molecules containing the SV40 origin also replicated to a significant extent (10 to 20% of circular molecules). Finally, electron microscopy of replication intermediates demonstrated that the initiation of DNA synthesis in vivo takes place at a unique site corresponding to the in vivo origin and that replication is bidirectional. These findings provide further evidence that replication in the cell-free system faithfully mimics SV40 DNA replication in vivo.     

Characterization of the replication of Escherichia coli DNA in the absence of protein synthesis: Stable DNA replication

After inhibiting DNA synthesis in Escherichia coli, repeated cycles of chromosome replication can occur in the absence of protein synthesis. This “stable” replication requires the products of all of the known dna genes.Stable replication results from inhibiting DNA synthesis by treatment with naladixic acid, cytosine arabinoside or hydroxyurea; or by placing dnaB, dnaE or dnaG mutants at non-permissive temperatures. It also follows a “shift-up” into rich medium in which RNA and protein are synthesized more rapidly than DNA. Paradoxically, stable replication is induced also by treatment with concentrations of streptolydigin which do not inhibit DNA replication but temporarily and partially inhibit RNA and protein synthesis. During all of these treatments, some protein synthesis must occur.Stable replication is not immediately expressed after a short period of thymine starvation or streptolydigin treatment, but requires a subsequent period of protein synthesis. Once established, however, the stable replication state is permanent and will persist in the absence of protein synthesis or during normal growth.After stable replication has been determined by a period of DNA inhibition, it is possible to inactivate replication by heating dnaA, B, C, E and G temperature-sensitive mutants. However, resynthesis of these gene products in the presence of thymine and at the permissive temperature restores stable replication activity. Since restoration of activity can occur under normal growth conditions which do not induce stable replication, it was concluded that the dnaA, B, C, E and G gene products do not directly determine the stabilized character of the replication fork.A model is presented which attempts to explain the ability of different treatments to induce stable replication.     

Inhibition of DNA replication in preimplantation mouse embryos by aphidicolin

The regulation of trophectoderm differentiation in mouse embryos was studied by inhibiting DNA synthesis with aphidicolin, a specific inhibitor of DNA polymerase alpha. Embryos were exposed to aphidicolin (0.5 micrograms/ml) for 16 h at various preimplantation stages and scored for their ability to form a blastocyst and develop beyond the blastocyst stage. Embryos were most sensitive to aphidicolin at the late 4-cell stage and became progressively less sensitive as they developed. Aphidicolin inhibited blastocyst formation by 70%, 100%, 77%, and 24% after treatment at the 2-cell, 4-cell, noncompacted 8-cell, and compacted 8-cell stages, respectively. Although the inhibitory effect of aphidicolin on blastocyst formation decreased markedly as 8-cell embryos underwent compaction, developmental capacity beyond the blastocyst stage was poor after treatment of either noncompacted or compacted 8-cell embryos. Treatment at the morula and early blastocyst stages was less harmful to embryos than treatment at earlier stages but reduced the number of trophoblast outgrowths by interfering with hatching. Autoradiographic analysis showed that during aphidicolin treatment, incorporation of 3H-thymidine was inhibited over 90% at all stages examined, indicating an inhibition of DNA synthesis. Because inhibition of blastocyst formation by aphidicolin decreased at the compacted 8-cell stage, we suggest that approximately the first half of the fourth DNA replication cycle is critical for subsequent blastocyst formation. Furthermore, the poor further development of blastocysts formed after aphidicolin treatment of compacted 8-cell embryos suggests that the DNA replication requirements for initial trophectoderm differentiation are distinct from requirements for further development of blastocysts in vitro.     

Purification of a cellular, double-stranded DNA-binding protein required for initiation of adenovirus DNA replication by using a rapid filter-binding assay.

A rapid and quantitative nitrocellulose filter-binding assay is described for the detection of nuclear factor I, a HeLa cell sequence-specific DNA-binding protein required for the initiation of adenovirus DNA replication. In this assay, the abundant nonspecific DNA-binding activity present in unfractionated HeLa nuclear extracts was greatly reduced by preincubation of these extracts with a homopolymeric competitor DNA. Subsequently, specific DNA-binding activity was detected as the preferential retention of a labeled 48-base-pair DNA fragment containing a functional nuclear factor I binding site compared with a control DNA fragment to which nuclear factor I did not bind specifically. This specific DNA-binding activity was shown to be both quantitative and time dependent. Furthermore, the conditions of this assay allowed footprinting of nuclear factor I in unfractionated HeLa nuclear extracts and quantitative detection of the protein during purification. Using unfrozen HeLa cells and reagents known to limit endogenous proteolysis, nuclear factor I was purified to near homogeneity from HeLa nuclear extracts by a combination of standard chromatography and specific DNA affinity chromatography. Over a 400-fold purification of nuclear factor I, on the basis of the specific activity of both sequence-specific DNA binding and complementation of adenovirus DNA replication in vitro, was affected by this purification. The most highly purified fraction was greatly enriched for a polypeptide of 160 kilodaltons on silver-stained sodium dodecyl sulfate-polyacrylamide gels. Furthermore, this protein cosedimented with specific DNA-binding activity on glycerol gradients. That this fraction indeed contained nuclear factor I was demonstrated by both DNase I footprinting and its function in the initiation of adenovirus DNA replication. Finally, the stoichiometry of specific DNA binding by nuclear factor I is shown to be most consistent with 2 mol of the 160-kilodalton polypeptide binding per mol of nuclear factor I-binding site.     

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

Summary 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 g/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.     

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.     

The Bacillus subtilis DnaD protein: a putative link between DNA remodeling and initiation of DNA replication

The Bacillus subtilis DnaD protein is an essential protein and a component of the oriC and PriA primosomal cascades, which are responsible for loading the main replicative ring helicase DnaC onto DNA. We present evidence that DnaD also has a global DNA architectural activity, assembling into large nucleoprotein complexes on a plasmid and counteracting plasmid compaction in a manner analogous to that recently seen for the histone-like Escherichia coli HU proteins. This DNA-remodeling role may be an essential function for initiation of DNA replication in the Gram +ve B. subtilis, thus highlighting DnaD as the link between bacterial nucleoid reorganization and initiation of DNA replication.     

大肠杆菌TorS/TorR二组分体应答蛋白TorR对DNA复制起始的影响

二组分体作为一种信号转导系统在细菌中普遍存在,能够感知外界环境变化并做出应答。细菌中CckA/CtrA、ArcA/ArcB和PhoP/PhoQ二组分体与DNA复制起始和细胞分裂相关,但目前还未见TorS/TorR二组分体对细胞周期及DNA复制影响的相关报道。大肠杆菌TorS/TorR二组分体能够监测细胞周围氧化三甲胺(Trimethylamine oxide, TMAO)的浓度变化,但其是否影响DNA复制起始呢?文章利用流式细胞仪检测了ΔtorS和ΔtorR突变体菌株的复制式样。结果发现,ΔtorS突变菌株每个细胞复制起始原点数目和倍增时间与野生型细胞一致,而ΔtorR突变菌株每个细胞复制起始原点数目多于野生型细胞,说明复制起始发生时间比野生型细胞早。但是过表达TorR蛋白或者共同表达TorS和TorR蛋白都不能使ΔtorR突变体表型恢复为野生型表型。而在野生型和ΔtorR突变细胞中过表达SufD蛋白能使复制起始提早发生,在ΔtorR和ΔsufD双突变细胞中复制起始延迟。所以,TorR可能通过改变sufD基因的表达来间接影响染色体复制起始。     

The inhibition of both initiation and elongation of adenovirus DNA replication by actinomycin D

The effect of actinomycin D on adenovirus DNA replication has been examined both in vivo and in a cell-free extract capable of replication on exogenously added template. In both cases we show that 5 micrograms/ml of drug cause an inhibition of DNA synthesis of at least 80%. The in vitro results further demonstrate that both DNA chain growth (elongation) and initiation - the addition of the first nucleotide of the DNA chain (dCMP) to the preterminal protein - are inhibited directly by the drug, by not by alpha-amanitin.