Studies on bacteriophage T7 DNA synthesis in vitro. II. Reconstitution of the T7 replication system using purified proteins.

Summary DNA synthesis in vitro using intact duplex T7 DNA as template is dependent on a novel group of three phage T7-induced proteins: DNA-priming protein (activity which complements a cell extract lacking the T7 gene 4-protein), T7 DNA polymerase (gene 5-protein plus host factor), and T7 DNA-binding protein. The reaction requires, in addition to the four deoxyribonucleoside triphosphates, all four ribonucleoside triphosphates and is inhibited by low concentrations of actinomycin D. Evidence is presented that the priming protein serves as a novel RNA polymerase to form a priming segment which is subsequently extended by T7 DNA polymerase. T7 RNA polymerase (gene 1-protein) can only partially substitute for the DNA-priming protein. At 30°C, deoxyribonucleotide incorporation proceeds for more than 2 hours and the amount of newly synthesized DNA can exceed the amount of template DNA by 10-fold. The products of synthesis are not covalently attached to the template and sediment as short (12S) DNA chains in alkaline sucrose gradients. Sealing of these fragments into DNA of higher molecular weight requires the presence of E. coli DNA polymerase I and T7 ligase. Examination of the products in the electron microscope reveals many large, forked molecules and a few eye-shaped structures resembling the early replicative intermediates normally observed in vivo.     

Bacteriophage-T7-induced DNA-priming protein. A novel enzyme involved in DNA replication.

The T7gene-4 protein has been purified to near homogeneity using a complementation assay in vitro, and it is designated T7 DNA-priming protein (DNA primase). The purified enzyme enables T7 DNA polymerase to initate DNA synthesis on various circular single-stranded DNA templates by a mechanism which involes the synthesis of a very short RNA primer. The oligoribonucleotide, which is linked to the product DNA via a 3':5'-phosphodiester bond, starts with pppA-C and terminates predominantly with AMP. When only ATP and CPT are precursors, the RNA primer is found to be primarily a tetranucleotide of the sequence pppA-C-C-A. Using oligoribonucleotides in place of ribonucleoside triphosphates as chain initators, T7 DNA-priming protein drastically increases the efficiency with which T7 DNA polymerase can utilize particular tetranucleotide primers containing A and C residues. T7 DNA-priming protein also enables T7 DNA polymerase to make use of native or nicked duplex T7 DNA as template-primer. This reaction does not require ribonucleoside triphosphates, although their addition enhances DNA synthesis 2--4 fold. The product formed in their absence is covalently attached to the template DNA and is found to contain a few long branches when examined by electron microscopy. In the presence of ribonucleoside triphosphates most of the newly made product arises from imitation of DNA chains de novo. Incubation of three proteins: T7 DNA-priming protein, T7 DNA polymerase, and T7 DNA-binding protein, with ribonucleoside and deoxyribonucleoside triphosphates, and with phiX174DNA as template leads to the generation of 'rolling circle-like' structures as visualized in the electron microscope. Single-stranded regions at the tail-circle junction indicate that initations can occur de novo on the displaced complementary strand. This is consistent with a discontinuous mode of 'lagging' strand synthesis and suggests that the same proteins may also be responsible for fork propagation in vivo.     

Replication of origin containing adenovirus DNA fragments that do not carry the terminal protein.

Nuclear extracts from adenovirus type 5 (Ad5) infected HeLa cells were used to study the template requirements for adenovirus DNA replication in vitro. When XbaI digested Ad5 DNA, containing the parental terminal protein (TP), was used as a template preferential synthesis of the terminal fragments was observed. The newly synthesized DNA was covalently bound to the 82 kD preterminal protein (pTP). Plasmid DNAs containing the Ad2 origin sequence or the Ad12 origin sequence with small deletions were analyzed for their capacity to support pTP-primed DNA replication. Circular plasmid DNAs were inactive. When plasmids were linearized to expose the adenovirus origin, both Ad2 and Ad12 TP-free fragments could support initiation and elongation similarly as Ad5 DNA-TP, although with lower efficiency. These observations indicate that the parental terminal protein is dispensable for initiation in vitro. The presence of 29 nucleotides ahead of the molecular end or a deletion of 14 base pairs extending into the conserved sequence (9-22) destroyed the template activity. DNA with a large deletion within the first 8 base pairs could still support replication while a small deletion could not. The results suggest that only G residues at a distance of 4-8 nucleotides from the start of the conserved sequence can be used as template during initiation of DNA replication.     

The involvement of RNA in the initiation of DNA synthesis in mammalian cells. Artifacts arising during caesium-salt density-gradient centrifugation which simulate a covalent attachment of RNA to newly synthesized DNA.

Artifacts were encountered during caesium salt density gradient centrifugation which simulated results expected if newly synthesized DNA is covalently attached to RNA. Newly synthesized DNA (in baby hamster kidney cells, BHK-21/C13), pulse-labelled with [3H]thymidine for 10 min at temperatures below 25 degrees C, banded at a greater buoyant density than mature [14C]DNA (heat-denatured nucleic acids) when centrifuged to equilibrium in caesium chloride gradients. Some of the newly synthesized RNA, labelled with [3H]uridine, banded at a buoyant density slightly greater than DNA in caesium sulphate gradients. These results were not obtained when nucleic acids were pulse-labelled at 37 degrees C, nor when samples were heat-denatured in the presence of formaldehyde. This suggests that nucleic acids can aggregate during centrifugation; this is discussed in relation to the molecular size of the DNA.     

Dissection of RNA-primed DNA synthesis catalyzed by gene 4 protein and DNA polymerase of bacteriophage T7. Coupling of RNA primer and DNA synthesis

Gene 4 protein and DNA polymerase of bacteriophage T7 catalyze RNA-primed DNA synthesis on single-stranded DNA templates. T7 DNA polymerase exhibits an affinity for both gene 4 protein and single-stranded DNA, and gene 4 protein binds stably to single-stranded DNA in the presence of dTTP (Nakai, H. and Richardson, C. C. (1986) J. Biol. Chem. 261, 15208-15216). Gene 4 protein-T7 DNA polymerase-template complexes may be formed in both the presence and absence of nucleoside 5'-triphosphates. The protein-template complexes may be isolated free of unbound proteins and nucleotides by gel filtration and will catalyze RNA-primed DNA synthesis in the presence of ATP, CTP, and the four deoxynucleoside 5'-triphosphates. RNA-primed DNA synthesis may be dissected into separate reactions for primer synthesis and DNA synthesis. Upon incubation of gene 4 protein with single-stranded DNA, ATP, and CTP, a primer-template complex is formed; it is likely that gene 4 protein mediates stable binding of the oligonucleotide to the template. The complex, purified free of unbound proteins and nucleotides, supports DNA synthesis upon addition of DNA polymerase and deoxynucleoside 5'-triphosphates. Association of primers with the template is increased by the presence of dTTP or DNA polymerase during primer synthesis. DNA synthesis supported by primer-template complexes initiates predominantly at gene 4 recognition sequences, indicating that primers are bound to the template at these sites.     

The control of chondroitin sulphate biosynthesis and its influence on the structure of cartilage proteoglycans.

Chondroitin sulphate synthesis on proteoglycans was decreased in rat chondrosarcoma cell cultures in the presence of cycloheximide (0.1-1.0 muM) or p-nitrophenyl beta-D-xyloside (50 microM). In the presence of cycloheximide the proteoglycan monomer was of larger size, the chondroitin sulphate chains were increased in length, but a similar number of chains was attached to each proteoglycan and the size of the core protein was unaltered. In the presence of p-nitrophenyl beta-D-xyloside (50 microM), chondroitin sulphate synthesis was increased (by 60-80%), but the incorporation into proteoglycans was decreased (by 70%). The chondroitin sulphate chains were of shorter length than in control cultured and the number of chains attached to each proteoglycan was decreased. In cultures with cycloheximide or actinomycin D the synthesis of chondroitin sulphate was less inhibited on beta-xyloside than on endogenous proteoglycan. When the rate of chondroitin sulphate synthesis was decreased by lowering the temperature of cultures, the chains synthesized at 22 and 4 degrees C were much longer than at 37 degrees C, but in the presence of p-nitrophenyl beta-D-xyloside the chains were of the same length at all three temperatures. A model of chain elongation is thus proposed in which the rate of chain synthesis is determined by the concentration of xylosyl acceptor and the length of the chains is determined by the ratio of elongation activity to xylosyl-acceptor concentration.     

Specific initiation of replication at the right-end telomere of the closed species of minute virus of mice replicative-form DNA.

We have developed an in vitro system that supports the replication of natural DNA templates of the autonomous parvovirus minute virus of mice (MVM). MVM virion DNA, a single-stranded molecule bracketed by short, terminal, self-complementary sequences, is converted into double-stranded replicative-form (RF) DNA when incubated in mouse A9 fibroblast extract. The 3' end of the newly synthesized complementary strand is ligated to the right-end hairpin of the virion strand, resulting in the formation of a covalently closed RF (cRF) molecule as the major conversion product. cRF DNA is not further replicated in A9 cell extract alone. On addition of purified MVM nonstructural protein NS1 expressed from recombinant baculoviruses or vaccinia viruses, cRF DNA is processed into a right-end (5' end of the virion strand) extended form (5'eRF). This is indicative of NS1-dependent nicking of the right-end hairpin at a distinct position, followed by unfolding of the hairpin and copying of the terminal sequence. In contrast, no resolution of the left-end hairpin can be detected in the presence of NS1. In the course of the right-end nicking reaction, NS1 gets covalently attached to the right-end telomere of the DNA product, as shown by immunoprecipitation with NS1-specific antibodies. The 5'eRF product is the target for additional rounds of NS1-induced nicking and displacement synthesis at the right end, arguing against the requirement of the hairpin structure for recognition of the DNA substrate by NS1. Further processing of the 5'eRF template in vitro leads to the formation of dimeric RF (dRF) DNA in a left-to-left-end configuration, presumably as a result of copying of the whole molecule by displacement synthesis initiated at the right-end telomere. Formation of dRF DNA is highly stimulated by NS1. The experimental results presented in this report support various assumptions of current models of parvovirus DNA replication and provide new insights into the replication functions of the NS1 protein.     

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.     

Initiation of adenovirus DNA replication: detection of covalent complexes between nucleotide and the 80-kilodalton terminal protein

We have previously shown that the 5'-terminal deoxycytidine residue of each nascent adenovirus 5 DNA strand synthesized in vitro is covalently linked to the 80-kilodalton (kd) terminal protein precursor via a phosphodiester bond to a serine residue in the protein. When extracts prepared from adenovirus 5-infected cells are incubated with [alpha-33P]dCTP as the only added deoxynucleoside triphosphate, complexes consisting of nucleotide covalently linked to the 80-kd protein can be detected. The nucleotide moieties present in such complexes include d(pC) and d(pCpA), the 5'-terminal nucleotide and dinucleotide of adenovirus 5 DNA, respectively, as well as some longer oligonucleotides. The formation of these complexes requires the presence of adenovirus DNA containing the attached 55-kd terminal protein and ATP. Extracts from H5ts125-infected cells which are defective in DNA replication catalyze complex formation to the same extent as extracts prepared from wild-type infected cells; thus, the presence of the adenovirus-coded 72-kd DNA-binding protein is apparently not required. Most, if not all, of the 80-kd protein-nucleotide complexes that are formed are noncovalently bound to the input viral DNA. These observations are consistent with the protein-priming model for the initiation of adenovirus DNA replication.     

Effects of chloramphenicol and rifampicin on the replication of R plasmid NR1 deoxyribonucleic acid in Escherichia coli.

The effects of inhibition of protein and ribonucleic acid (RNA) synthesis on the replication of the plasmids NR1 and F′lac in Escherichia coli were studied. When protein synthesis is inhibited, there is approximately a 25% increase in R plasmid deoxyribonucleic acid (DNA), but this newly synthesized DNA is not recoverable in the covalently closed circular (CCC) form until protein synthesis is allowed to resume. When RNA synthesis is inhibited, there is also approximately a 20% increase in R plasmid DNA, but this DNA is immediately recoverable in the CCC form. F′lac DNA, unlike R plasmid DNA, can continue to replicate for at least a generation time in the absence of protein synthesis, and this F′lac DNA is immediately recoverable in the CCC form.     

De novo initiation of RNA synthesis by the RNA-dependent RNA polymerase (NS5B) of hepatitis C virus

Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn(2+) than in the presence of Mg(2+). When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a "copy-back" mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3' end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (>/=50 microM) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo.     

An oxidized nucleotide affects DNA replication through activation of protein kinases in Xenopus egg lysates

To elucidate the response to oxidative stress in eukaryotic cells, the effect of an oxidized nucleotide, 8-oxo-2′-deoxyguanosine 5′-triphosphate (8-oxo-dGTP), generated from dGTP with an active oxygen, on DNA synthesis was studied using a cell-free DNA replication system derived from Xenopus egg lysates with a single-stranded DNA template. Amounts of newly synthesized DNA were reduced according to the increasing concentration of 8-oxo-dGTP. Pulse labeling analysis revealed that 8-oxo-dGTP could delay DNA synthesis by reducing the rate of chain elongation. This delay was recovered by addition of a protein kinase inhibitor, staurosporine or bisindolylmaleimide I. These results indicate that a staurosporine- or bisindolylmaleimide I-sensitive protein kinase, such as a protein kinase C family member, may contribute to the delay of DNA synthesis by 8-oxo-dGTP. UV-irradiated single-stranded DNA also caused a delay of DNA synthesis on the undamaged template in the lysates. However, this delay was not recovered by staurosporine or bisindolylmaleimide I. Therefore, the mechanism of delay of DNA synthesis by 8-oxo-dGTP may be different from that by UV lesions. This is the first report that demonstrates an effect of an oxidized nucleotide on DNA replication in eukaryotes.     

Characteristics of deoxyribonucleic acid polymerase activity in nuclear and supernatant fractions of cultured mouse cells

1. DNA polymerase activity is present in both nuclear and supernatant fractions prepared from rapidly dividing L929 mouse cells. 2. Nuclear preparations are 2-5 times more active with added native DNA as template and the supernatant fractions show an equivalent preference for heat-denatured DNA. 3. Isolated nuclei can carry on limited DNA synthesis in the absence of added template but are stimulated five- to ten-fold by addition of 50mug of native DNA per assay. 4. DNA polymerase activity can be released from intact nuclei by ultrasonic treatment or by extraction with 1.5m-potassium chloride. 5. The activities in nuclear and supernatant fractions, with their preferred templates, respond similarly to changes in pH and Mg(2+) and K(+) concentrations. 6. Maximal enzyme activity is approached with 40mug of DNA per assay and activation of the DNA template by treatment with deoxyribonuclease does not decrease the amount of DNA required to reach saturation. 7. The nuclear enzyme, incubated with native DNA, is markedly inhibited by the addition of heat-denatured DNA to the assay. In contrast, the supernatant DNA polymerase activity on denatured templates is not affected by the presence of native DNA. 8. The nuclear enzyme exhibits high activity in the absence of one or more deoxyribonucleoside triphosphates but this is much diminished after partial purification of the enzyme by precipitation at pH5 and fractionation on Sephadex G-200 columns. 9. The (3)H-labelled DNA products formed by Sephadex-purified nuclear and supernatant fractions, with their preferred templates, were found to be resistant to treatment with exonuclease I. Alkali-denaturation of the (3)H-labelled DNA products rendered them susceptible to attack by exonuclease I. 10. Analysis of the products on alkaline sucrose density gradients suggests that the newly synthesized material may not be covalently bound to the original DNA template. 11. By using their preferred templates the specific activity of supernatant fractions varies markedly with the position of the cells in the cell-cycle, but the specific activity of nuclear fractions varies only slightly.     

Ribonucleotides associated with a gap in newly replicated kinetoplast DNA minicircles from Trypanosoma equiperdum

In Trypanosoma equiperdum, some newly replicated kinetoplast DNA minicircles contain a single gap at a unique location in their newly synthesized strand (Ntambi, J. M., and Englund, P. T. (1985) J. Biol. Chem. 260, 5574-5579). We now report that ribonucleotides are associated with this gap, with one or two covalently attached to the 5' terminus of the newly synthesized strand. There appear to be two possible RNA/DNA junctions at adjacent positions in the sequence. The ribonucleotides may be remnants of a replication primer, and their presence strongly implies that the gap is at the site of a replication origin.     

Mechanism of inhibition of deoxyribonucleic acid synthesis by 1-beta-D-arabinofuranosyladenosine triphosphate and its potentiation by 6-mercaptopurine ribonucleoside 5'-monophosphate

The mechanism of inhibition of DNA synthesis by 1-beta-D-arabinofuranosyl-ATP (ara-ATP) and the potentiation of this inhibition by 6-mercaptopurine ribonucleoside 5'-monophosphate (6-MPR-P) have been investigated with mammalian DNA polymerase delty by using poly(dA-dT) as the template. The inhibition of DNA synthesis by ara-ATP correlates with incorporation of ara-AMP into poly(dA-dT). Nearest-neighbor analysis indicates that ara-AMP does not act as an absolute chain terminator but rather that chains with 3'-terminal arabinosyl nucleotides are extended slowly. The inhibition of DNA synthesis by ara-ATP is markedly enhanced by the addition of the nucleotide derivative of 6-mercaptopurine, 6-mercaptopurine ribonucleoside 5'-monophosphate. The increased inhibition of DNA synthesis in the presence of 6-MPR-P is due to increased incorporation of ara-AMP. The mechanism by which 6-MPR-P increases the incorporation of ara-AMP is by selective inhibition of the 3' to 5' exonuclease activity of DNA polymerase, thereby preventing the removal of newly incorporated ara-AMP at 3' termini of DNA chains.     

Influence of template primary and secondary structure on the rate and fidelity of DNA synthesis

High resolution gel electrophoresis was used to monitor the successive addition of dNMP residues onto the 3'-OH ends of discrete 5'-32P-primers, during DNA synthesis on natural templates. Resulting autoradiographic banding patterns revealed considerable variation in the relative rates of incorporation at different positions along the template. The pattern of "pause sites" along the template was unique for each of three different DNA polymerases (polymerase I (the "large fragment" form of Escherichia coli), T4 polymerase (encoded by bacteriophage T4), and AMV polymerase (DNA polymerase of avian myeloblastosis virus]. Most pause sites were not caused by attenuation of polymerization at regions of local secondary structure in the template. Assays of the accuracy of incorporation at different positions along the template (in which elongation was monitored in the presence of only 3 of the 4 2'-deoxynucleoside 5'-triphosphates) strongly suggested that the relative fidelity of DNA synthesis catalyzed by different polymerases depends on the position on the template at which the comparison is made. Primer-templates were constructed that permitted comparison of elongation during synthesis on a single-stranded template with that during polymerization through a double-stranded region (wherein elongation required concomitant displacement of a strand annealed adjacent to the 5'-32P-primer). Although strand displacement DNA synthesis catalyzed by polymerase I occurred approximately ten times more slowly than synthesis in the same region of a single-stranded viral template, most of the pause sites were the same in the presence or absence of "tandem" primer. Electrophoretic assays of the fidelity of DNA synthesis suggested that an increased tendency toward misincorporational "hotspots" occurred when elongation required concomitant strand displacement.