依赖于DNA的RNA聚合酶与体外转录的研究 Ⅰ.酵母变异株20B-12 RNA聚合酶A和C的纯化及鉴定

从酵母变异株20B-12经过超声波处理、硫酸铵沉淀、DEAE纤维素和磷酸纤维素层析等步骤,纯化依赖于DNA的RNA聚合酶A和C,得到聚丙烯酰胺凝胶电泳均一的条带。其中不含DNase、RNase 和蛋白酶活力,无内源DNA。测定了 RNA 聚合酶A和C对α-鹅膏荤碱的敏感性。酶A在α-鹅膏荤碱为400μg/ml时,活性受到抑制,而酶C在该浓度时,几乎不受抑制。(NH_4)_2SO_4对酶A的最适浓度为20mM,对酶C有二个最适浓度,分别为40mM和240mM。无二价金属离子Mn~(2+)或Mg~(2+),酶A和C几乎无活力。两种酶最适Mn~(2+)浓度均为2.5mM,Mg~(2+)浓度均为5mM。两种酶以热变性小牛胸腺DNA为模板,测活性均较天然小牛胸腺DNA为模板时高。     

The adenovirus DNA binding protein and adenovirus DNA polymerase interact to catalyze elongation of primed DNA templates

The adenovirus-encoded 140-kDa DNA polymerase (Ad Pol) and the 59-kDa DNA binding protein (Ad DBP) are both required for the replication of viral DNA in vivo and in vitro. Previous studies demonstrated that, when poly(dT).oligo(dA) was used as a template-primer, both proteins were required for poly(dA) synthesis. In this report, the interaction between the Ad Pol and Ad DBP was further investigated using poly(dT).oligo(dA) as well as a linear duplex molecule containing 3' poly(dT) tails. DNA synthesis with the tailed template required Ad Pol, Ad DBP, and an oligo(dA) primer hydrogen bonded to the poly(dT) tails. Incorporation was stimulated 8-10-fold by ATP; however, no evidence of ATP hydrolysis to ADP was observed. Synthesis was initiated at either end of the tailed molecule and proceeded through the duplex region to the end of the molecule. This ability to translocate through duplex DNA and to synthesize long poly(dA) chains suggests that the Ad Pol.Ad DBP complex can act efficiently in the elongation reactions involved in the replication of Ad DNA (both type I and type II). During the replication reaction, substantial hydrolysis of deoxynucleoside triphosphates to the corresponding deoxynucleoside monophosphates occurred. This reaction required DNA synthesis and most likely reflects an idling reaction similar to that observed with other DNA polymerases containing 3'----5' exonuclease activity in which the polymerase first incorporates and then hydrolyzes a dNMP.     

Cellular RNA synthesis in normal and mengovirus-infected L-929 cells.

Cellular RNA synthesis was studied in mouse L-929 cells and in these cells infected with mengovirus. RNA polymerases I, II, and III were partially purified and their chromatographic properties were analyzed by DEAE-Sephadex A-25 chromatography. RNA polymerase II was purified from mouse liver and its subunit structure was compared to that of normal and virus-infected L-929 cells by two-dimensional gel electrophoresis. By these criteria, the enzymes from all three sources were identical. The RNA synthetic activities and capacities of chromatins from normal and virus-infected cells were compared under a variety of conditions. The endogenous activity in chromatin from infected cells was inhibited relative to controls but the residual activity responded normally to stimulation by ammonium sulfate, heparin, and Sarkosyl. The template capacity of the chromatins was compared with added RNA polymerase II and by a rifampicin challenge assay utilizing Escherichia coli RNA polymerase. Identical results were obtained in each case. The number of growing RNA chains and the rates of their elongations were determined. The results showed that nuclei and chromatin from infected cells have a smaller number of RNA polymerase II molecules engaged in RNA synthesis than normal cells do but that the active molecules elongate RNA chains at the same rate.     

Origin of adenovirus DNA replication. Role of the nuclear factor I binding site in vivo

An assay is described that detects in vivo a single round of initiation and DNA synthesis directed by a linear molecule containing an exposed single copy of an adenovirus (Ad) origin of replication. This and a previously described assay, which measures multiple rounds of DNA replication, were used to identify DNA sequences within the Ad2 and Ad4 origins of replication that are important for ori function. Linear DNA molecules containing sequences from the Ad2 or Ad4 genome termini were cotransfected with homologous and heterologous helper virus, and net amounts of DNA synthesis were compared. Linear molecules containing the Ad4 inverted terminal repeats were replicated 20-fold better in the presence of the homologous helper, whereas both Ad2 and Ad4 inverted terminal repeats were utilized efficiently by Ad4. DNA sequence analysis of the Ad2 ori and the corresponding region in Ad4 indicated that, although there are only ten variant base-pairs, eight are located within the Ad2 DNA sequence recognized by the cellular protein nuclear factor I. This protein is required to achieve the maximal rate of Ad2 DNA replication in vitro, and these differences therefore identify DNA sequences that are crucial to Ad2 ori function. The Ad4 ITR does not contain a functional nuclear factor I binding site, and deletion analysis has demonstrated that this region of the Ad4 genome is not required for ori function. In contrast to Ad2, the DNA sequences required for the initiation of Ad4 DNA replication were shown to reside entirely within the terminal 18 base-pairs of the Ad4 inverted terminal repeat.     

The DNA polymerases associated with the adenovirus type 2 replication complex: effect of 2''-3''-dideoxythymidine-5''-triphosphate on viral DNA synthesis.

An adenovirus (Ad) DNA replication complex extracted from infected HeLa nuclei could be purified free of the bulk of intracellular DNA polymerase activity by sedimetation in neutral sucrose gradients. However, the replication complex still retained some alpha and gamma DNA-polymerase activity. Since this complex is inhibited by 2', 3' dideoxythymidine-5'-triphosphate (ddTTP), an inhibitor of DNA polymerase gamma, a functional role for this enzyme in Ad DNA replication is suggested. Similar inhibition by ddTTP in intact Ad infected nuclei and comparable inhibition of Ad DNA synthesis in whole cells by dideoxythymidine (ddThy) are consistent with a role for DNA polymerase gamma. Uninfected HeLa nuclei or whole cells are not similarly inhibited by ddTTP or DDThy respectively. Such data does not rule out an additional functional role for other DNA polymerases, and recent experiments from this laboratory (1) suggest that DNA polymerase alpha is also involved in Ad DNA synthesis.     

Functional interaction between Epstein-Barr virus DNA polymerase catalytic subunit and its accessory subunit in vitro.

The Epstein-Barr virus (EBV) DNA polymerase catalytic subunit (BALF5 protein) and its accessory subunit (BMRF1 protein) have been independently overexpressed and purified (T. Tsurumi, A. Kobayashi, K. Tamai, T. Daikoku, R. Kurachi, and Y. Nishiyama, J. Virol. 67:4651-4658, 1993; T. Tsurumi, J. Virol. 67:1681-1687, 1993). In an investigation of the molecular basis of protein-protein interactions between the subunits of the EBV DNA polymerase holoenzyme, we compared the DNA polymerase activity catalyzed by the BALF5 protein in the presence or absence of the BMRF1 polymerase accessory subunit in vitro. The DNA polymerase activity of the BALF5 polymerase catalytic subunit alone was sensitive to high ionic strength on an activated DNA template (80% inhibition at 100 mM ammonium sulfate). Addition of the polymerase accessory subunit to the reaction greatly enhanced DNA polymerase activity in the presence of high concentrations of ammonium sulfate (10-fold stimulation at 100 mM ammonium sulfate). Optimal stimulation was obtained when the molar ratio of BMRF1 protein to BALF5 protein was 2 or more. The DNA polymerase activity of the BALF5 protein along with the BMRF1 protein was neutralized by a monoclonal antibody to the BMRF1 protein, whereas that of the BALF5 protein alone was not, suggesting a specific interaction between the BALF5 protein and the BMRF1 protein in the reaction. The processivity of nucleotide polymerization of the BALF5 polymerase catalytic subunit on singly primed M13 single-stranded DNA circles was low (approximately 50 nucleotides). Addition of the BMRF1 polymerase accessory subunit resulted in a strikingly high processive mode of deoxynucleotide polymerization (> 7,200 nucleotides). These findings strongly suggest that the BMRF1 polymerase accessory subunit stabilizes interaction between the EBV DNA polymerase and primer template and functions as a sliding clamp at the growing 3'-OH end of the primer terminus to increase the processivity of polymerization.     

Adeno-associated virus DNA replication complexes in herpes simplex virus or adenovirus-infected cells.

A complex which is active in in vitro synthesis of adeno-associated virus (AAV) DNA was solubilized from Vero cells that were co-infected with AAV and either adenovirus (Ad5) or a herpes simplex virus type 1 (HSV-1) as the helper virus. The complexes from the Ad5 and HSV-1-infected cells sedimented at 23 S and 28 S, respectively. The optimal conditions for in vitro DNA synthesis for the two types of complex using the endogenous AAV template and the endogenous DNA polymerase, differed with respect to the effect of KCl and K2SO4 concentration. In addition the complex from HSV-1-infected cells, but not that from Ad5-infected cells, was inhibited by phosphonoacetic acid. Thus, the two complexes appear to contain different DNA polymerase activities. This was verified by phosphocellulose chromatography of the DNA polymerases solubilized from the isolated complexes. The major activity in the complex from HSV-1 infected cells was the HSV-induced DNA polymerase with lesser amounts of cellular DNA polymerase alpha and gamma or both. The complex from the Ad5-infected cells contained mainly a cellular DNA polymerase gamma.     

Structural analysis of viral replicative intermediates isolated from adenovirus type 2-infected HeLa cell nuclei.

Deoxyribonucleoprotein complexes released 17 h postinfection from adenovirus type 1 (Ad2)-infected HeLa cell nuclei were shown by electron microscopy to contain filaments much thicker (about 200 A [20 nm]) than double-stranded DNA (about 20 A [2 nm]). The complexes were partially purified through a linear sucrose gradient, concentrated, and further purified in a metrizamide gradient. The major protein present in the complexes was identified as the 72,000-dalton (72K), adenovirus-coded single-stranded DNA-binding protein (72K DBP). Three types of complexes have been visualized by electron microscopy. Some linear complexes were uniformly thick, and their length corresponded roughly to that of the adenovirus genome. Other linear genome-length complexes appeared to consist of a thick filament connected to a thinner filament with the diameter of double-stranded DNA. Forked complexes consisting of one thick filament connected to a genome-length, thinner double-stranded DNA filament were also visualized. Both thick and thin filaments were sensitive to DNase and not to RNase, but only the thick filaments were digested by the single-strand-specific Neurospora crassa nuclease, indicating that they correspond to a complex of 72K DBP and Ad2 single-stranded DNA. Experiments with anti-72K DBP immunoglobulins indicated that these nucleoprotein complexes, containing the 72K DBP, correspond to replicative intermediates. Both strands of the Ad2 genome were found associated to the 72K DBP. Altogether, our results establish the in vivo association of the 72K DBP with adenovirus single-stranded DNA, as previously suggested from in vitro studies, and support a strand displacement mechanism for Ad2 DNA replication, in which both strands can be displaced. In addition, our results indicate that, late in infection, histones are not bound to adenovirus DNA in the form of a nucleosomal chromatine-like structure.     

Suppressor cell activity in tumor-bearing mice. III. Co-purification of a factor inhibiting cellular DNA synthesis and DNA polymerase activity

The factor(s) derived from fibrosarcoma-induced suppressor T cells was sensitive to pronase and neuraminidase, but not to trypsin, beta-galactosidase, DNase, or RNase. Protein and RNA, but not DNA, synthesis were required to mediate suppression. Suppressor T cell-derived factor(s) could be precipitated by a 50% saturated ammonium sulfate (SAS) solution. The 50% SAS fraction inhibited both in vitro and in vivo spleen cell blastogenesis, whereas the 80% and unprecipitated fractions had no inhibitory activity. Using Sephadex G-200 chromatography, the 2nd protein fraction (fraction II) contained an inhibitor of both DNA polymerases (IDP) and DNA synthesis (IDS) activity, which possessed no cytotoxic activity. In vitro DNA polymerase alpha activity was suppressed by fraction II, whereas DNA polymerase beta and gamma activities remained unchanged. Molecular weight of IDP/IDS, as determined by Sephadex G-200 gel filtration chromatography, was approximately 14,500. Attempts to separate IDP/IDS activities found in fraction II by anion-exchange chromatography and slab gel electrophoresis were not successful, which suggested that the 2 activities were the same or very similar molecules.     

Purification and functional characterization of adenovirus ts111A DNA-binding protein. Fluorescence studies of protein-nucleic acid binding

The adenovirus single-stranded DNA (ssDNA)-binding protein (DBP) is necessary for the elongation step in viral DNA replication. In an attempt to characterize the putative ssDNA-binding domain of the DBP, we purified and characterized the Ad2ts111A DBP, which contains a glycine-to-valine substitution at amino acid 280. This mutation is adjacent to that in the previously studied Ad2+ND1ts23. Ad2+ND1ts23 exhibits a temperature-sensitive defect in DNA replication, and its DBP has previously been shown to bind ssDNA with reduced affinity. Ad2ts111A DBP, like Ad2+ND1ts23, does not support adenovirus DNA replication in vitro at elevated temperatures. However, the Ad2ts111A DBP binds ssDNA more tightly than does Ad2+ND1ts23 and is not temperature sensitive in this function. To determine the nucleic acid-binding properties of DBP, we applied spectrofluorometric techniques, which had not been used previously to study adenovirus DBP. Using the homopolynucleotide poly(1,N6)-ethenoadenylic acid (poly(r epsilon A], we have determined that the binding site size is approximately 16 nucleotides. In 20 mM NaCl, the Ad2wt, Ad2ts111A, and Ad2+ND1ts23 DBP proteins all bound stoichiometrically to poly(r epsilon A) with overall apparent affinities above 108 M-1. Based on titrations carried out at higher salt concentrations, however, the stability of these complexes did appear to increase in the order Ad2+ND1ts23 less than Ad2ts111A less than Ad2wt. By these techniques, we have confirmed also that the DBP of another temperature-sensitive mutant, H5ts107, like the Ad2ts111A DBP, retains its ability to bind ssDNA even at a restrictive temperature utilizing the salt concentration compatible with adenovirus DNA replication in vitro. The H5ts107 DBP, which contains an amino acid substitution at position 413, is defective for in vitro replication at nonpermissive temperature but is not temperature sensitive for binding to ssDNA. In summary, our results indicate that the replication defects of the Ad2ts111A are similar to those of H5ts107 and cannot be attributed to defective, nonspecific ssDNA binding by the DBP. It appears that ssDNA binding by itself is not sufficient to account for the role of DBP in adenovirus 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.     

Replication of simian virus 40 chromatin in vitro depends on the amount of DNA polymerase alpha associated with replicating chromatin

Replication of simian virus 40 (SV40) chromatin in vitro is inhibited by chloride but stimulated by acetate anions even at physiological concentrations of 100-200 mM. In a similar fashion DNA polymerase alpha is affected with respect to the activity with activated DNA as primer template. Furthermore, at concentrations of 100-200 mM acetate DNA polymerase alpha remains associated with replicating chromatin, whereas association is strongly reduced when chloride anions are used at the corresponding concentrations. Thus the salt behaviour of DNA polymerase alpha explains the salt sensitivity of the replication system. Our results confirm the importance of this enzyme for DNA replication.     

The role of exonucleolytic processing and polymerase-DNA association in bypass of lesions during replication in vitro. Significance for SOS-targeted mutagenesis

The role of exonuclease activity in trans-lesion DNA replication with Escherichia coli DNA polymerase III holoenzyme was investigated. RecA protein inhibited the 3'----5' exonuclease activity of the polymerase 2-fold when assayed in the absence of replication and had no effect on turnover of dNTPs into dNMPs. In contrast, single-stranded DNA-binding protein, which had no effect on the exonuclease activity in the absence of replication, showed a pronounced 7-fold suppression of the 3'----5' exonuclease activity during replication. The excision of incorporated dNMP alpha S residues from DNA by the 3'----5' exonuclease activity of DNA polymerase III holoenzyme was inhibited 10-20-fold; still no increase in bypass of pyrimidine photodimers was observed. Thus, in agreement with our previous results in which the exonuclease activity was inhibited at the protein level (Livneh, Z. (1986) J. Biol. Chem. 261, 9526-9533), inhibition at the DNA level also did not increase bypass of photodimers. Fractionation of the replication mixture after termination of DNA synthesis on a Bio-Gel A-5m column under conditions which favor polymerase-DNA binding yielded a termination complex which could perform turnover of dNTPs into dNMPs. Adding challenge-primed single-stranded DNA to the complex yielded a burst of DNA synthesis which was promoted most likely by DNA polymerase III holoenzyme molecules transferred from the termination complex to the challenge DNA thus demonstrating the instability of the polymerase-DNA association. Addition of a fresh sample of DNA polymerase III holoenzyme to purified termination products, which consist primarily of partially replicated molecules with nascent chains terminated at UV lesions, did not result in any net DNA synthesis as expected. However, reactivation of lesion-terminated primers was achieved by pretreatment with a 3'----5' exonuclease which excised 200 nucleotides or more, generating new 3'-OH termini located away from the UV lesions. When these exonuclease-treated products were subjected to a second round of replication, an increased level of DNA synthesis was observed including additional bypass of photodimers. These results suggest the possibility that 3'----5' exonuclease processing might be required at least transiently during one of the stages of trans-lesion DNA replication, which is believed to be the mechanism of SOS-targeted mutagenesis.     

Adenoviruses with nonidentical terminal sequences are viable.

Adenovirus genomes consist of linear DNA molecules containing inverted terminal repeat sequences (ITRs) of 100 to 200 base pairs. The importance of identical termini for viability of adenoviruses was investigated. The viral strains used in this study were wild-type adenovirus type 5 (Ad5) and a variant Ad2 strain with termini which were distinct from those of all other human adenoviruses sequenced to date. A hybrid virus (sub54), obtained by recombination between Ad2 and Ad5, derived the left 42 to 52% of its genome from Ad2 and the right 58 to 48% from Ad5. Southern blotting analysis with labeled oligodeoxynucleotides indicated that both Ad2 and Ad5 ITRs were present in sub54 viral DNA preparations, and successive plaque purifications of sub54 demonstrated that viruses with nonidentical terminal sequences were viable but were rapidly converted to viruses with identical ends. Cloning of the sub54 genome as a bacterial plasmid supported the observations made by analysis of sub54 virion DNA. A plasmid, pFG154, was isolated which contained the entire adenovirus genome with an Ad2 ITR at the left terminus covalently linked to an Ad5 ITR at the right terminus. Upon transfection of mammalian cells with pFG154, viral progeny were obtained which had all possible combinations of termini, thus confirming that molecules with nonidentical termini are viable. Pure populations of viruses with nonidentical termini could not be isolated, suggesting efficient repair of one end with the opposite terminus used as a template. A model for this process is proposed involving strand displacement replication and emphasizing the importance of panhandle formation (annealing of terminal sequences) as a replicative intermediate.     

A simplified procedure for the analysis of DNA polymerase III levels in Bacillus subtilis strains.

A simple and reproducible procedure is described which allows the fast and almost quantitative removal of DNA polymerases I and II from DNA polymerase III, in crude extracts of polA+ strains of Bacillus subtilis. The procedure entails streptomycin sulfate and ammonium sulfate fractionations; subsequent analysis of the partially purified preparation by G-200 chromatography, DEAE cellulose chromatography and density gradient sedimentation, shows that the ammonium sulfate fraction contains less than 5% of the total activity as DNA polymerase I and less than 2% as DNA polymerase II. The purification procedure, up to the ammonium sulfate step, was utilized for the analysis of the level of DNA polymerase III in several B. subtilis mutants, with results comparable to those obtained from the corresponding polA- strains following more cumbersome purification procedures. The M.W. of the purified form is of 227.000, somewhat greater than the published values. The early fractions of the purification have revealed the existence of a form with a M.W. of 426.000; the nature of this form, which has been observed in several instances and which is very unstable and short-lived, is under investigation.     

Interactions of the DNA polymerase and gene 4 protein of bacteriophage T7. Protein-protein and protein-DNA interactions involved in RNA-primed DNA synthesis

Three proteins catalyze RNA-primed DNA synthesis on the lagging strand side of the replication fork of bacteriophage T7. Oligoribonucleotides are synthesized by T7 gene 4 protein, which also provides helicase activity. DNA synthesis is catalyzed by gene 5 protein of the phage, and processivity of DNA synthesis is conferred by Escherichia coli thioredoxin, a protein that is tightly associated with gene 5 protein. T7 DNA polymerase and gene 4 protein associate to form a complex that can be isolated by filtration through a molecular sieve. The complex is stable in 50 mM NaCl but is dissociated by 100 mM NaCl, a salt concentration that does not inhibit RNA-primed DNA synthesis. T7 DNA polymerase forms a stable complex with single-stranded M13 DNA at 50 mM NaCl as measured by gel filtration, and this complex requires 200 mM NaCl for dissociation, a salt concentration that inhibits RNA-primed DNA synthesis. Gene 4 protein alone does not bind to single-stranded DNA. In the presence of MgCl2 and dTTP or beta, gamma-methylene dTTP, a gene 4 protein-M13 DNA complex that is stable at 200 mM NaCl is formed. The affinity of DNA polymerase for both gene 4 protein and single-stranded DNA leads to the formation of a gene 4 protein-DNA polymerase-M13 DNA complex even in the absence of nucleoside triphosphates. However, the binding of each protein to DNA plays an important role in mediating the interaction of the proteins with each other. High concentrations of single-stranded DNA inhibit RNA-primed DNA synthesis by diluting the amount of proteins bound to each template and reducing the frequency of protein-protein interactions. Preincubation of gene 4 protein, DNA polymerase, and M13 DNA in the presence of dTTP forms protein-DNA complexes that most efficiently catalyze RNA-primed DNA synthesis in the presence of excess single-stranded competitor DNA.