Studies on the Mechanism of Replication of Adenovirus DNA II. The Nature of Single-Stranded DNA in Replicative Intermediates

Replicative intermediates of adenovirus type 5 DNA contain large stretches of single-stranded DNA. We have shown that this single-stranded DNA is mainly of parental origin, whereas all new DNA synthesized during one round of replication has a double-stranded structure. Hybridization experiments of the single-stranded DNA with isolated complementary strands of adenovirus type 5 DNA showed that this DNA hybridized only with the viral L-strand (the strand with the lower equilibrium density in alkaline CsCl) indicating that it represents the viral H-strand. This observation implies that replication always starts from one and the same molecular end. Electron microscopy of partially denatured Y-shaped intermediates confirmed this and showed that replication started from the molecular right end (the end richest in A-T base pairs). In conclusion, we have shown that replication of adenovirus type 5 DNA starts at the molecular right end, displacing the parental H-strand.     

Complementary strands of CELO virus DNA.

When alkali-denatured DNA from CELO virus (an avian adenovirus) was annealed for 15 min at 37 C in 0.1 M NaCl, 70% of the molecules formed single-stranded circles. This is probably due to base pairing of complementary sequences not more than 110 nucleotides long at the ends of the single strands and implies an inverted terminal repetition in the duplex DNA similar to that reported for the DNA from human adenoviruses. The circular molecules had a uniform length that was approximately the same as that of linear single-stranded molecules. The complementary strands of CELO virus DNA were separated on a preparative scale, and at least 40% of the heavy strands and 56% of the light strands were found to be intact as judged by the formation of single-stranded circles.     

Relationship of mRNA from Productively Infected Cells to the Complementary Strands of Adenovirus Type 2 DNA

The complementary strands of adenovirus type 2 (Ad2) DNA were separated by buoyant density gradient centrifugation with poly (U, G). The complementary strand DNA was shown to remain intact through the course of strand separation. The l-strand of Ad2 DNA, appearing in the less dense complex with poly (U, G) in neutral CsCl density gradients, was shown to have a buoyant density in alkaline (pH 12.5) CsCl density gradients which is 2 to 3 mg per ml greater than that of its complement (h-strand). Renaturation of purified complementary strand DNA was observed only in mixtures of h- and l-strand DNA, and then with the second-order reaction rate expected for Ad2 DNA. Hybridization of the complementary strands of Ad2 DNA with cytoplasmic mRNA isolated from infected HeLa cells was performed in liquid phase and analyzed by hydroxylapatite chromatography. Before viral DNA synthesis (6 h after infection), 13 to 18% of the h-strand and 30 to 35% of the l-strand were represented in viral mRNA. Late (18 h) after infection the mRNA represented 20 to 25% and 63 to 68% of the h- and l-strands, respectively. Most, if not all sequences present in viral mRNA before viral DNA synthesis were also present in the cytoplasm late in infection.     

Transcription map for adenovirus type 12 DNA.

The regions of the adenovirus type 12 genome which encode l- and r-strand-specific cytoplasmic RNA were mapped by the following procedure. Radioactive, intact, separated complementary strands of the viral genome were hybridized to saturating amounts of unlabeled late cytoplasmic RNA. The segments of each DNA strand complementary to the RNA were then purified by S1 nuclease digestion of the hybrids. The arrangement of the coding regions of each strand was deduced from the pattern of hybridization of these probes to unlabeled viral DNA fragments produced by digestion with EcoRI, BamHI, and HindIII.. The resulting map is similar, if not identical, to that of adenovirus type 2. The subset of the late cytoplasmic RNA sequences which are expressed at early times were located on the map by hybridizing labeled, early cytoplasmic RNA to both unlabeled DNA fragments and unlabeled complementary strands of specific fragments. Early cytoplasmic RNA hybridized to the r-strand to EcoRI-C and BamHI-B and to the l-strand of BamHI-E. Hybridization to BamHI-C was also observed. The relative rates of accumulation of cytoplasmic RNA complementary to individual restriction fragments was measured at both early and late times. Early during infection, most of the viral RNA appearing in the cytoplasm was derived from the molecular ends of the genome. Later (24 to 26 h postinfection) the majority of the newly labeled cytoplasmic RNA was transcribed from DNA sequences mapping between 25 and 60 map units on the genome.     

Two complementary strand-specific termination sites for adenovirus DNA replication

Adenovirus type 2 DNA, specifically labeled at the termini for DNA replication, was prepared by isolation of viral DNA molecules which were completed during short pulses with ³H-thymidine. The distribution of radioactivity in the two complementary strands at the termini for DNA replication was determined by liquid phase hybridization and gel electrophoresis. At the right-hand terminus, nearly all radioactivity was found in the viral h strand, whereas at the left-hand terminus, most radioactivity was confined to the viral I strand. The results suggest that both molecular ends serve as origins and termini for replication of adenovirus type 2 DNA.     

Nature of the single-stranded DNA in replicating adenovirus type 5 DNA.

Single-stranded regions in replicating adenovirus type 5 DNA were isolated and hybridized in solution to the separated strands of adenovirus 2 or 5 DNA. The results showed that the two strands of adenovirus 5 DNA are exposed to almost the same extent during replication, suggesting that displacement synthesis may start from either end of the viral DNA.     

Complementary strand-specific sequences from unique fragments of adenovirus type 2 DNA for hybridization-mapping experiments

Separation of the complementary strands of adenovirus type 2 DNA by poly(U,G)-CsCl density gradient centrifugation permitted studies of Ad2³ DNA renaturation with independently variable concentrations of each complementary strand. Single-stranded DNA was isolated by hydroxylapatite chromatography following exhaustive incubation under such conditions, and was found to selectively represent sequences of the complement present in excess during the incubation. This result was exploited in a general method for isolation of complementary strand-specific sequences of radioactively labeled Ad2 DNA or restriction enzyme fragments of Ad2 DNA. Liquid phase saturation-hybridization experiments were carried out with labeled DNA representing each complementary strand of the six endo R.EcoRI cleavage fragments of Ad2 DNA and unlabeled messenger RNA prepared from HeLa cells late after productive infections with Ad2. The results were combined with the known endo R.EcoRI cleavage map of Ad2 DNA to construct a low-resolution map showing physically separated regions, on both complementary strands of Ad2 DNA, represented in mRNA late after infection.     

Termination sites for adenovirus type 2 DNA replication.

Termination sites for replication of adenovirus type 2 DNA have been demonstrated at both ends of the viral chromosome by the procedure of Danna and Nathans (1972). Single-stranded DNA from replicating intermediates was also characterized by hybridization with separated strands of viral DNA. The results indicate that both strands are exposed during replication.     

Comparison of viral RNA sequences in adenovirus 2-transformed and lytically infected cells

The complementary strands of fragments of ³²P-labelled adenovirus 2 DNA generated by cleavage with restriction endonucleases EcoRI or Hpa1 were separated by electrophoresis. Saturation hybridization reactions were performed between these fragment strands and unlabelled RNA extracted from the cytoplasm of adenovirus 2-transformed rat embryo cells or from human cells early after adenovirus 2 infection. The fraction of each fragment strand complementary to RNA from these sources was measured by chromatography on hydroxylapatite. Maps of the viral DNA sequences complementary to messenger RNA in different lines of transformed cells and early during lytic infection of human cells were constructed.Five lines of adenovirus 2-transformed cells were examined. All contained the same RNA sequences, complementary to about 10% of the light strand of EcoRI fragment A. DNA sequences coding for this RNA were more precisely located using Hpa1 fragments E and C and mapped at the left-hand end of the genome. Thus any viral function expressed in all adenovirus 2-transformed cells, tumour antigen, for example, must be coded by this region of the viral genome. Two lines, F17 and F18, express only these sequences; two others, 8617 and REM, also contain mRNA complementary to about 7% of the heavy strand of the right-hand end of adenovirus 2 DNA; a fifth line, T2C4, contains these and many additional viral RNA sequences in its cytoplasm.The viral RNA sequences found in all lines of transformed cells are also present in the cytoplasm of human cells during the early phase of a lytic adenovirus infection. The additional cytoplasmic sequences in the 8617 and REM cell lines also correspond to “early” RNA sequences.     

A single-stranded DNA-binding protein of bacteriophage T7 defective in DNA annealing

The annealing of complementary strands of DNA is a vital step during the process of DNA replication, recombination, and repair. In bacteriophage T7-infected cells, the product of viral gene 2.5, a single-stranded DNA-binding protein, performs this function. We have identified a single amino acid residue in gene 2.5 protein, arginine 82, that is critical for its DNA annealing activity. Expression of gene 2.5 harboring this mutation does not complement the growth of a T7 bacteriophage lacking gene 2.5. Purified gene 2.5 protein-R82C binds single-stranded DNA with a greater affinity than the wild-type protein but does not mediate annealing of complementary strands of DNA. A carboxyl-terminal-deleted protein, gene 2.5 protein-Delta26C, binds even more tightly to single-stranded DNA than does gene 2.5 protein-R82C, but it anneals homologous strands of DNA as well as does the wild-type protein. The altered protein forms dimers and interacts with T7 DNA polymerase comparable with the wild-type protein. Gene 2.5 protein-R82C condenses single-stranded M13 DNA in a manner similar to wild-type protein when viewed by electron microscopy.     

DNA strand exchange and selective DNA annealing promoted by the human immunodeficiency virus type 1 nucleocapsid protein.

Nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) was expressed in Escherichia coli and purified. The protein displayed a variety of activities on DNA structure, all reflecting an ability to promote transition between double-helical and single-stranded conformations. We found that, in addition to its previously described ability to accelerate renaturation of complementary DNA strands, the HIV-1 NC protein could substantially lower the melting temperature of duplex DNA and could promote strand exchange between double-stranded and single-stranded DNA molecules. Moreover, in the presence of HIV-1 NC, annealing of a single-stranded DNA molecule to a complementary DNA strand that would yield a more stable double-stranded product was favored over annealing to alternative complementary DNA strands that would form less stable duplex products (selective annealing). NC thus appears to lower the kinetic barrier so that double-strand <==> single-strand equilibrium is rapidly reached to favor the lowest free-energy nucleic acid conformation. This activity of NC may be important for correct folding of viral genomic RNA and may have practical applications.     

Rifampicin-resistant initiation of DNA synthesis on the isolated strands of ColE plasmid DNA.

The opposite strands of the ColE1 and ColE3 plasmids were isolated as circular single-stranded DNA molecules. These molecules were compared with M13 and phi X174 viral DNA with respect to their capacity to function as templates for in vitro DNA synthesis by a replication enzyme fraction from Escherichia coli. It was found for both ColE plasmids that the conversion of H as well as L strands to duplex DNA molecules closely resembles phi X174 complementary strand synthesis and occurs by a rifampicin-resistant priming mechanism involving the dnaB, dnaC, and dnaG gene products. Restriction analysis of partially double-stranded intermediates indicates that preferred start sites for DNA synthesis are present on both strands of the ColE1 HaeII-C fragment. Inspection of the nucleotide sequence of this region reveals structural similarities with the origin of phi X174 complementary strand synthesis. We propose that the rifampicin-resistant initiation site (rri) in the ColE1 L strand is required for the priming of discontinuous lagging strand synthesis during vegetative replication and that the rri site in the H strand is involved in the initiation of L strand synthesis during conjugative transfer.     

Reassociation of complementary strand-specific adenovirus type 2 DNA with viral DNA sequences of transformed cells.

Complementary strand-specific adenovirus DNA, either full length or from restriction enzyme cleavage fragments, was used to estimate the fractional representation and abundance of viral sequences in two adenovirus type 2 (Ad2)-transformed rat cell lines, A2F19 and A2T2C4. The reassociation method introduced is based on the linear relationship, after exhaustive hybridization, between the inverted fraction of hybrid DNA and the molar ratio of probe to cellular DNA in the reaction mixture. The amount of viral DNA in A2F19 cells represents 12 to 14% of the viral genome at a level of around seven copies per diploid cell equivalent. For the cell line A2T2C4, the pattern of integrated viral DNA sequences is more complex. With full-length Ad2 DNA strands as a probe, about 56% of the probe was represented in cellular DNA. When each of the four BamHI fragment strands of Ad2 DNA was used as a probe, the fraction of the viral DNA present also amounted to around 56% with one to five copies from different regions of the viral genome. The results demonstrate the advantage of using strand-specific viral DNA as a probe in reassociation analysis with denatured cell DNA. The method should be useful in any system in which complementary strand separation of viral DNA sequences can be achieved.     

Transcriptional organization of the Drosophila melanogaster ribosomal RNA genes.

Five distinct DNA replicating intermediates have been separated from lysates of bacteriophage G4-infected cells pulse-labelled during the period of replicative form synthesis using propidium diiodide/caesium chloride gradients. These are a partially single-stranded theta structure that is labelled in both the viral and complementary DNA strands; partially single-stranded circles, some with an unfinished viral DNA strand (25%) and some with an unfinished complementary DNA strand (75%); replicative form II(RFII) and replicative form I(RFI) DNA labelled only in the complementary DNA strand. To explain the pulse-label data a model is proposed in which G4 replicative form replication takes place by a displacement mechanism in which synthesis of the new viral DNA strand displaces the old viral DNA strand as a single-stranded DNA loop (D-loop) and when the displacement reaches half way round the molecule (the origin of synthesis of the G4 viral and complementary DNA strands are on opposite sides of the genome, Martin &; Godson 1977) synthesis of the complementary DNA strand starts, but in the opposite direction. Strand separation of the parent helix runs ahead of DNA synthesis, releasing two partially single-stranded circles from the replicating structure which then complete their replication as free single-stranded DNA circles. No evidence was found to support a rolling circle displacement mechanism of G4 replicative form synthesis.     

Type I DNA topoisomerases from mammalian cell nuclei interlock strains and promote renaturation of denatured closed circular PM2 DNA

Type I DNA topoisomerases from mouse ascites cell nuclei and from rat liver cell nuclei act on denatured viral closed circular PM2 DNA to produce molecules with a highly contracted structure as well as fully duplex non-supercoiled covalently closed circular molecules. Highly contracted DNA molecules contain a novel type of topological linkage in which a strand in one region of the double-stranded molecule passes between the strands in another region of the circular molecule one or more times. Since it is also found that the action of the topoisomerase promotes renaturation of complementary strands in denatured closed circular DNA, it is suggested that formation of contracted DNA structures proceeds through renatured, duplex intermediates with highly negative superhelix densities that contain small single-stranded regions.     

Renaturation of complementary DNA strands by herpes simplex virus type 1 ICP8.

ICP8, the major single-stranded DNA-binding protein of herpes simplex virus type 1, promotes renaturation of complementary single strands of DNA. This reaction is ATP independent but requires Mg2+. The activity is maximal at pH 7.6 and 80 mM NaCl. The major product of the reaction is double-stranded DNA, and no evidence of large DNA networks is seen. The reaction occurs at subsaturating concentrations of ICP8 but reaches maximal levels with saturating concentrations of ICP8. Finally, the renaturation reaction is second order with respect to DNA concentration. The ability of ICP8 to promote the renaturation of complementary single strands suggests a role for ICP8 in the high level of recombination seen in cells infected with herpes simplex virus type 1.     

Networks of DNA and RecA protein are intermediates in homologous pairing

Partial coating of single-stranded DNA by recA protein causes its aggregation, but conditions that promote complete coating inhibit independent aggregation of single strands and, instead, cause the mutually dependent conjunction of single- and double-stranded DNA in complexes that sediment at more than 10 000 S. This coaggregation is independent of homology but otherwise shares key properties of homologous pairing of single strands with duplex DNA: both processes require ATP, MgCl2, and stoichiometric amounts of recA protein; both are very sensitive to inhibition by salt and ADP. Coaggregates are closed domains that are intermediates in homologous pairing: they form faster than joint molecules, they include virtually all of the DNA in the reaction mixture, and they yield joint molecules nearly an order of magnitude faster than they exchange DNA molecules with the surrounding solution. The independent aggregation of single-stranded DNA differs in all respects except the requirement for Mg2+, and its properties correlate instead with those associated with the renaturation of complementary single strands by recA protein.     

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.     

Letter: Some unusual properties of replicating adenovirus type 2 DNA

Replicating adenovirus type 2 DNA was isolated from KB cells 13 hours after infection. The buoyant density in caesium chloride of the replicating DNA was found to be 5 to 10 mg/cm² greater than that of mature adenovirus type 2 DNA. The single-strand specific nuclease from Neurospora crassa released 25 to 30% of the radioactivity from replicating DNA and the density difference between replicating and mature adenovirus DNA was eliminated after digestion with this enzyme, but not after digestion with RNase or pronase. The results suggest that the complementary strands of adenovirus type 2 DNA are replicated asynchronously.     

Autonomous parvovirus LuIII encapsidates equal amounts of plus and minus DNA strands.

Autonomous parvoviruses are thought to uniquely encapsidate single-stranded DNA of minus polarity. In contrast, the defective adeno-associated viruses separately encapsidate equal amounts of plus and minus DNA strands. We reexamined the uniqueness of minus strand encapsidation for the autonomous parvoviruses. Although we found that Kilham rat virus and H-1 virus encapsidate varying but small amounts of complementary-strand DNA, it was unexpected to find that LuIII virus encapsidated equal amounts of plus and minus DNA. The extracted LuIII DNA possessed properties of double-stranded replicative-form DNA, including insensitivity to S1 endonuclease, cleavage by restriction enzymes, and conversion to unit-length, single-stranded DNA when electrophoresed under denaturing conditions. However, the inability of this DNA to form single-stranded DNA circles when denatured and then renatured in the presence of formamide and the lack of double-stranded DNA circle formation after treatment with exonuclease III and reannealing shows a lack of sequence homology of the 3' and 5' termini of LuIII DNA, in contrast to adeno-associated virus DNA. Digestion of LuIII double-stranded DNA with EcoRI and HincII and separation of plus and minus DNA strands on composite agarose-acrylamide gels identified a heterogeneity present only in the plus DNA strand. These results suggest that strand specificity of viral DNA encapsidation is not a useful property for differentiation between the autonomous and defective parvoviruses. Furthermore, encapsidation by LuIII of equal amounts of complementary DNA strands in contrast to encapsidation of minus strands by H-1 virus, when propagated in the same host cell type, suggests that selection of strands for encapsidation is a virus-coded rather than host-controlled event.