Replication of Bacteriophage Ribonucleic Acid: Some Properties of Native and Denatured Replicative Intermediate

Purified replicative form (RF) and replicative intermediate (RI) prepared from Escherichia coli cells infected with the ribonucleic acid (RNA) bacteriophage R17 were denatured with dimethyl sulfoxide at 37 C or in aqueous solvents of low ionic strength at 97 C. Denaturation was demonstrated for RF and RI by an increase in specific infectivity and a striking change in the hyperchromicity curves after treatment. RI denaturation was also demonstrated by a shift in the buoyant density in Cs(2)SO(4) from 1.619 to the buoyant density of single-stranded R17 RNA (1.627). Analysis of the denatured RI hyperchromicity curves and the equilibrium distributions of denatured RI in Cs(2)SO(4) gradients revealed, however, a residual double-stranded component. Velocity sedimentation of denatured RI was performed, and the weight distribution of S values was calculated. From the known relation between molecular weight and S values, it was possible to transform the weight distribution into a number distribution of chain lengths. This distribution was compared with that predicted from the steady-state hypothesis for RI. Deviations from the predicted distribution may be due to the residual double-stranded component.     

Denaturation and Renaturation of Viral Ribonucleic Acid I. Annealing R17 Ribonucleic Acid with Denatured Replicative Form or with Denatured Replicative Intermediate

Purified replicative form (RF) and replicative intermediate (RI) prepared from Escherichia coli infected with R17 were denatured in 0.15 m NaCl, 0.015 m sodium citrate containing 65% dimethylsulfoxide. Denaturation of RF or RI was demonstrated spectrophotometrically, chromatographically, and by sedimentation analysis. Denatured RF or RI was annealed by carefully decreasing the temperature from 62 to 20 C. Annealing was accompanied by a decreased absorbance at 260 mmu. The decrease in absorbance during annealing appeared to be dependent upon the rate of cooling and the concentration of ribonucleic acid (RNA). Denatured RF or RI was annealed with R17 RNA which was labeled with (3)H-uridine. The annealed product was 73 to 82% resistant to 0.1 mug/ml of ribonuclease. Annealing R17 RNA with either denatured RF or RI resulted in the formation of a ribonuclease-resistant product with a sedimentation profile resembling that of native RI. Melting the annealed products in 85.7% dimethyl sulfoxide produced 27S single-stranded R17 RNA and a heterogeneous population of more slowly sedimenting RNA.     

Replication process of the parvovirus H-1. VII. Electron microscopy of replicative-form DNA synthesis.

The geometry of replicative form (RF) DNA synthesis of the H-1 parvovirus was studied with the electron microscope using formamide or aqueous variations of the Kleinschmidt spreading procedure. H-1 DNA was isolated from human or hamster cells infected with a temperature-sensitive mutant, ts1, which is deficient in progeny single-stranded DNA synthesis at the restrictive temperature (S.L. Rhode, 1976), thus minimizing possible confusion between RF and progeny DNA replicative intermediates (RIs). The purity of the isolated H-1 DNA, as determined by gel electrophoresis, ethidium bromide staining, autoadiography, and digestion with endo R-EcoRI, was high. H-1 RF DNA'S WERE LINEAR DOUBLE-STRANDED MOLECULES, 1.53 MUM IN LENGTH. H-1 RIs of RF DNA replication were double-stranded, Y-shaped molecules, with the same length as RF DNAs. The replication origin was localized no more than 0.15 genome lengths from one end of the RF DNA, with replication proceeding toward the other end at a uniform rate. Similar RF and RI molecules of dimer size were also observed. The length of H-1 single-stranded DNA extracted from purified virions was measured relative to that of phiX174 and it had a very similar contour length, so that the molecular weight of H-1 single-stranded DNA would be at least 1.48 X 10(6) to 1.59 X 10(6) (Berkowitz and Day, 1974).     

Chromatography of Arbovirus Ribonucleic Acid Forms on Columns of Benzoylated-Diethylaminoethyl Cellulose

Benzoylated-diethylaminoethyl cellulose (BD-cellulose) column chromatography was found to be useful in resolving most of the ribonucleic acid (RNA) forms from the replicative cycle of group A arbovirus Semliki Forect virus (SFV). The elution patterns were independent of molecular weight and appeared to be related to the degree of secondary structure in the molecule. Fractions of RNA were taken from a sucrose density gradient of cytoplasmic extracts of SFV-infected chick cells pretreated with actinomycin D. In a linear salt gradient, 16S material cochromatographed with the rapidly eluted ribonuclease resistant core of the double-stranded SFV-RNA and with the homopolymer duplex polyinosinic acid: polycytidylic acid. This fraction, therefore, probably contains an SFV-RNA form similar to the completely double stranded replicative form (RF) of several RNA viruses and bacteriophages. Faster moving (>20S) sucrose gradient fractions eluted more slowly, suggesting a decreasing proportion of secondary structure with increasing sedimentation value. The fractions, therefore, seemed to contain replicative intermediate (RI) structures. The two single stranded forms of SFV-RNA (42S and 26S) could only be eluted from BD-cellulose in the presence of urea or dimethyl sulfoxide, suggesting the presence of minimal secondary structure. Under these conditions, the single-stranded viral RNA forms could not be resolved. Molecular sieve chromatography of the single-stranded RNA forms, performed by passage through an agarose column, also failed to resolve these forms. The viral RNA forms containing a high degree of secondary structure, probably the RF and the RI, could, therefore, be rapidly separated from each other and from the single-stranded forms.     

Structure of poliovirus replicative intermediate RNA: Electron microscope analysis of RNA cross-linked in vivo with psoralen derivative

The structure of the poliovirus replicative intermediate RNA was examined by electron microscopy after cross-linking in vivo with 4′-aminomethyl-4,5′,8-trimethylpsoralen. After purification from infected cells, undenatured RI² appeared as a double-stranded backbone of genome length, with an average of three (and occasionally up to eight) nascent, single-stranded tails. After denaturation, however, only single strands of heterogeneous length were visualized, indicating that the RI in the cell contains little or no duplex structure, and thus nascent chains are only transiently hydrogen-bonded to their template over short regions. The double-stranded backbone of undenatured RI, observed previously by others and in these experiments, is due to collapse of complementary chains during the deproteinization and purification procedures. The effectiveness of the in vivo cross-linking procedure was demonstrated by the complete inhibition of viral RNA synthesis in treated cells and by direct binding of [³H]AMT to RI molecules in vivo. Mature polio virions are impermeable to AMT; however, growth of virus in cells incubated with AMT in the dark resulted in normal yields of virus particles containing RNA genomes, whose infectivity could be subsequently photo-inactivated. The frequency of AMT-induced cross-linking was determined by analyses of double-stranded poliovirus RNA (RF). Cross-linking in vitro followed by spreading for electron microscopy under denaturing conditions yielded bubbled duplex structures with a minimum of one interstrand cross-link per 80 base-pairs. RF cross-linked in vivo also showed extensive cross-linking, decreased about fivefold from the in vitro cross-linked value. Thus, the failure to detect cross-linked RI under these conditions indicates that extensive base-pairing does not exist in vivo.     

Structure and Function of Bacteriophage R17 Replicative Intermediate Ribonucleic Acid II. Properties of the Parental Labeled Molecule

Replicative intermediate ribonucleic acid (RNA), designated RI, which contained parental RNA labeled with (32)P was separated by filtration through agarose from the nucleic acids prepared from (32)P-labeled RNA phage-infected Escherichia coli. A larger amount of ribonuclease-sensitive parental label was found in the rapidly sedimenting forms of RI than in the slower sedimenting forms, indicating that parental RNA is displaced to form a single-stranded tail. This result indicates that some phage RNA is generated by asymmetric semiconservative replication of RI, but it does not mean that a portion of the RI duplexes cannot be conserved during generation of phage RNA. Parental RNA was also found in double-stranded RNA with no apparent tails which sedimented with an S value of 13. This RNA was soluble in 2 m NaCl, and its sedimentation rate was unaffected by ribonuclease; nevertheless, single-strand scissions were produced by ribonuclease and were detected after the duplex was converted to its component single strands.     

Synthesis of Replicative Form Deoxyribonucleic Acid and Messenger Ribonucleic Acid by Gene IV Mutants of Bacteriophage S13

Gene IV mutants of bacteriophage S13 are known to be blocked in infectious replicative form (RF) DNA synthesis, producing only a small fraction of the RF formed by wild-type phage. This investigation shows that gene IV mutants form only parental RF and are blocked in the synthesis of any progeny RF, either infectious or noninfectious. This was determined by density labeling of RF in cells treated with mitomycin C to suppress host deoxyribonucleic acid (DNA) synthesis. RF synthesis was also studied in untreated cells, using methylated albumin columns to separate RF from host DNA. In this case it was also found that synthesis of progeny RF by gene IV mutants is negligible. It has been found by DNA-ribonucleic acid (RNA) hybridization experiments that gene IV mutants form at least as much or more messenger RNA than wild-type phage. Therefore, parental RF alone can form messenger RNA in appreciable amounts.     

Replicative DNA Polymerases

In 1959, Arthur Kornberg was awarded the Nobel Prize for his work on the principles by which DNA is duplicated by DNA polymerases. Since then, it has been confirmed in all branches of life that replicative DNA polymerases require a single-stranded template to build a complementary strand, but they cannot start a new DNA strand de novo. Thus, they also depend on a primase, which generally assembles a short RNA primer to provide a 3′-OH that can be extended by the replicative DNA polymerase. The general principles that (1) a helicase unwinds the double-stranded DNA, (2) single-stranded DNA-binding proteins stabilize the single-stranded DNA, (3) a primase builds a short RNA primer, and (4) a clamp loader loads a clamp to (5) facilitate the loading and processivity of the replicative polymerase, are well conserved among all species. Replication of the genome is remarkably robust and is performed with high fidelity even in extreme environments. Work over the last decade or so has confirmed (6) that a common two-metal ion-promoted mechanism exists for the nucleotidyltransferase reaction that builds DNA strands, and (7) that the replicative DNA polymerases always act as a key component of larger multiprotein assemblies, termed replisomes. Furthermore (8), the integrity of replisomes is maintained by multiple protein–protein and protein–DNA interactions, many of which are inherently weak. This enables large conformational changes to occur without dissociation of replisome components, and also means that in general replisomes cannot be isolated intact.The genomes, from the smallest to the largest, provide an enormous challenge for the replicative DNA polymerases to faithfully copy to give the many generations that follow a comparable condition for life. In this article, we discuss the structural and functional bases by which replicative DNA polymerases are able to efficiently and faithfully build new copies of genomes in eubacteria, archaea, and eukaryotes.     

Denaturation and Renaturation of Viral Ribonucleic Acid II. Characterization of the Products Resulting from Annealing R17 Ribonucleic Acid with Denatured Replicative Form or with Denatured Replicative Intermediate

The ribonucleic acid (RNA) product resulting from annealing R17 RNA with denatured replicative form or replicative intermediate could be divided into two distinct types of RNA by precipitation in 1.5 m NaCl. The RNA found in the salt supernatant fluid was resistant to digestion by ribonuclease, had a sedimentation coefficient of 15S, and displayed a sharp thermal transition. The RNA in the salt supernatant fluid appeared to be identical to replicative form. The RNA found in the salt precipitate was resistant to digestion by ribonuclease, but possessed both single- and double-stranded characteristics. The RNA sedimented as a broad band in a sucrose gradient, with a sedimentation coefficient of 15S, and displayed a melting transition characteristic of a mixture of single- and double-stranded RNA. Mild ribonuclease digestion of the salt-precipitable RNA produced a ribonuclease-resistant material with sedimentation properties identical to the RNA found in the salt supernatant fluid.     

Studies on biosynthesis of tobacco mosaic virus. VII. Radioactivity of plus and minus strands in different forms of viral RNA after labelling of infected tobacco leaves

Tobacco leaves were labelled with tritiated undine for 30 or 120 minutes at different times after systemic infection with tobacco mosaic virus. RNA was extracted and separated into three fractions: one enriched in RF (replicative form), one enriched in RI (replicative intermediate), and one containing the bulk of single-stranded RNA. Radioactivity in plus strands (viral RNA) and minus strands (complementary RNA) was determined in each fraction by an isotope dilution assay. The amount of minus strands in the RP and RI fractions and the amount of plus strands in the single-stranded RNA fraction were also determined.Minus-strand synthesis was twice as high a few hours after the outbreak of visible symptoms as during the subsequent large accumulation of plus strands. At the early stage of virus production, the specific radioactivity of the minus strands was three- to fourfold that of the total RNA. Later it was about the same as that of the total RNA. As minus strands constitute a constant part of the total RNA at the later stages, this observation suggests that breakdown of minus strands is small.The specific radioactivity of minus strands was the same in corresponding RF and RI fractions. As the turn-over of minus strands appears to be small, a rapid interconversion of the two RNA types is indicated.In RF and RI the radioactivity in plus strands was between 6 and 50 times greater than that in minus strands. The specific radioactivity of plus strands was greater in RF and RI than in the single-stranded RNA, supporting the concept that both RF and RI have a precursor role for viral RNA.     

DNA Products in In Vitro DNA Synthesis Using Bacteriophage ϕX174 Single-stranded DNA

We described product analysis of DNA synthesized in chloroplast lysate from liverwort Marchantia polymorpha L. cell suspension cultures. Characteristics of in vitro DNA synthesis by chloroplast lysate using bacteriophage ?X174 single-stranded DNA were very similar to those in the case of double-stranded calf thymus DNA reported previously. Autoradiographic analysis clearly showed the incorporation of radioactive [α-³²P]-dCTP into DNA molecules associated with bacteriophage ?X174 single-stranded template DNA, indicating conversion of bacteriophage ?X174 single-stranded DNA to double-stranded DNA (RF III, double-stranded linear molecule). Experiments on the fate of [³²P]-labeled single-stranded DNA also showed a clear conversion of the single-stranded DNA to double-stranded DNA. Furthermore, patterns of sucrose density gradient centrifugations (neutral and alkaline) showed the production of two major components in in vitro DNA synthesis by chloroplast lysate. This also indicated conversion of bacteriophage ?X174 single-stranded DNA to double-stranded DNA (RF III form). Our results suggest that the mechanism of chloroplast DNA replication could be the mode of strand-displacement DNA synthesis as seen in animal mitochondrial DNA synthesis.     

DNA Structure Specificity Conferred on a Replicative Helicase by Its Loader

Prokaryotic and eukaryotic replicative helicases can translocate along single-stranded and double-stranded DNA, with the central cavity of these multimeric ring helicases being able to accommodate both forms of DNA. Translocation by such helicases along single-stranded DNA results in the unwinding of forked DNA by steric exclusion and appears critical in unwinding of parental strands at the replication fork, whereas translocation over double-stranded DNA has no well-defined role. We have found that the accessory factor, DnaC, that promotes loading of the Escherichia coli replicative helicase DnaB onto single-stranded DNA may also act to confer DNA structure specificity on DnaB helicase. When present in excess, DnaC inhibits DnaB translocation over double-stranded DNA but not over single-stranded DNA. Inhibition of DnaB translocation over double-stranded DNA requires the ATP-bound form of DnaC, and this inhibition is relieved during translocation over single-stranded DNA indicating that stimulation of DnaC ATPase is responsible for this DNA structure specificity. These findings demonstrate that DnaC may provide the DNA structure specificity lacking in DnaB, limiting DnaB translocation to bona fide replication forks. The ability of other replicative helicases to translocate along single-stranded and double-stranded DNA raises the possibility that analogous regulatory mechanisms exist in other organisms.     

In Vitro Synthesis of Poliovirus Ribonucleic Acid: Role of the Replicative Intermediate

Poliovirus ribonucleic acid (RNA) polymerase crude extracts could be stored frozen in liquid nitrogen without loss of activity or specificity. The major in vitro product of these extracts was viral single-stranded RNA. However, after short periods of incubation with radioactive nucleoside triphosphates, most of the incorporated label was found in replicative intermediate. When excess unlabeled nucleoside triphosphate was added, the label was displaced from the replicative intermediate and accumulated as viral RNA. It is concluded from this experiment that the replicative intermediate is the precursor to viral RNA. In addition, some of the label was chased into double-stranded RNA. The implications of this finding are discussed.     

Bacteriophage G4 DNA synthesis in temperature-sensitive dna mutants of Escherichia coli.

The synthesis of bacteriophage G4 DNA was examined in temperature-sensitive dna mutants under permissive and nonpermissive conditions. The infecting single-stranded G4 DNA was converted to the parental replicative form (RF) at the nonpermissive temperature in infected cells containing a temperature sensitive mutation in the dnaA, dnaB, dnaC, dnaE, or dnaG gene. The presence of 30 mug of chloramphenicol or 200 mug of rifampin per ml had no effect on parental RF synthesis in these mutants. Replication of G4 double-stranded RF DNA occurred at a normal rate in dnaAts cells at the nonpermissive temperature, but the rate was greatly reduced in cells containing a temperature-sensitive mutation in the dnaB, dnaC, dnaE, or dnaG gene. RF DNA replicated at normal rates in revertants of these dna temperature-sensitive host cells. The simplest interpretation of these observations is that none of the dna gene products tested is essential for the synthesis of the complementary DNA strand on the infecting single-stranded G4 DNA, whereas the dnaB, dnaC, dnaE, (DNA polymerase III), and dnaG gene products are all essential for replication of the double-stranded G4 RF DNA. The alternate possibility that one or more of the gene products are actually essential for G4 parental RF synthesis, even though this synthesis is not defective in the mutant hosts, is also discussed.     

Heterologous transfection with bacteriophage phiX174 DNA. Unusual heterogeneous products.

Hydroxylamine-resistant infectious materials (HARIM) synthesized in natural non-host and progeny phage low productive bacterial spheroplasts upon transfection with bacteriophage phiX174 DNA were found to be unusually heterogeneous in their forms. Using Pseudomonas aeruginosa as a source of HARIM, it was shown that they have the following unusual features. (1) Almost all of the HARIM are denser than normal single-stranded (SS)- and double-stranded replicative form (RF)-DNAs of phiX174 found usually in the phage-infected host cells. (2) A great part of these heavy HARIM (approximately 84%) contain a variable length of single-stranded RNA associated with their infectious elements. (3) For most of the HARIM (approximately 80% of total molecules as the infectious elements of the heavy HARIM), the infectious elements are phiX-RFI-DNA. The wide-spread system for phiX-HARIM synthesis was shown to be present in many gram-negative bacterial cells.     

Mechanism of viral replication. I. Structure of replication complexes of R17 bacteriophage

The replication of R17 bacteriophage in Escherichia coli MRE-600 cells was investigated using a new electron microscopic technique. The structures of replicating ribonucleoprotein complexes, as well as of purified replicative intermediate and replicative form, were studied. These structures are identical to those predicted by the model of Weissmann et al. (1968). From this it may be concluded that replication proceeds through essentially single-stranded inter-mediates and that double-stranded structures are either by-products or artifacts.     

Resistance of bacterial protein synthesis to double-stranded RNA

Double-stranded RNA fails to inhibit the formation of translation initiation complexes on R17 bacteriophage RNA, overall synthesis of R17 proteins, or the ability of bacterial initiation factor IF-3 to prevent the association of 30S and 50S ribosomal subunits into single ribosomes. Yet, IF-3 can form complexes with double-stranded RNA. However, IF-3 binds to double-stranded RNA with lower apparent affinity than to either R17 RNA or 30S ribosomal subunits; this may explain the resistance of bacterial protein synthesis to double-stranded RNA.     

Analysis of replicative intermediates produced during bacteriophage phi 29 DNA replication in vitro.

Replication of bacteriophage phi 29 DNA initiates at either end of its linear double-stranded DNA molecule and proceeds by a strand-displacement mechanism. In the present paper we have used an in vitro phi 29 DNA replication system to analyse by electron microscopy the replicative intermediates produced at different reaction times. Two types of replicative intermediates were observed: type I (full-length double-stranded phi 29 DNA molecules with one or more single-stranded DNA branches) and type II (full-length phi 29 DNA molecules formed by a double-stranded DNA portion of variable length from one end plus a single-stranded DNA portion spanning to the other end). Thus, the types of replicative intermediates produced in vivo were also formed in the in vitro phi 29 DNA replication system. Analysis of type I intermediates indicated that initiation of DNA replication occurs preferentially at both ends of the same DNA template, in a non-simultaneous manner. Type II intermediates appeared as early as two minutes after the reaction started, well before unit-length single-stranded phi 29 DNA molecules were synthesized. In addition, replication of recombinant phi 29 DNA templates lacking terminal protein at one end did not produce type II intermediates and led to an accumulation of full-length single-stranded phi 29 DNA molecules. These two observations strongly suggest that type II intermediates appear when two growing DNA chains, running from opposite ends, merge.