Construction of viable and lethal mutations in the origin of bacteriophage 'phi' X174 using synthetic oligodeoxyribonucleotides

Six different synthetic deoxyhexadecamers complementary to the origin of bacteriophage φX174, corresponding to nucleotides 4299 to 4314, except for one preselected nucleotide change were used as primers for DNA synthesis on wild-type φX² DNA as a template. DNA synthesis was performed with Escherichia coli DNA polymerase I (Klenow fragment) in the presence of DNA ligase. Heteroduplex RFIV DNA was isolated and, after limited digestion with DNAase I, complementary strands containing the mutant primers were isolated. The biological activity of these complementary strands was assayed in spheroplasts. Spheroplasts were made from E. coli K58 ung^? (uracil N-glycosylase) to prevent degradation of the complementary strands caused by uracil incorporation (Baas et al., 1980a).Using (5′-³²P) end-labeled primers, it was shown that all tested DNA polymerase preparations, including phage T4 DNA polymerase, contained variable amounts of 5′ → 3′ exonuclease activity. This nick translation activity may result in removal of the mutation in the primers, and therefore in isolation of wild-type complementary DNA instead of mutant complementary DNA.Restriction enzyme analysis of completed RFIV DNA showed that the primers can initiate DNA synthesis at more than one place on the φX174 genome. These complications result in a mixed population of complementary strand DNAs synthesized in vitro. Nevertheless, the desired mutants were picked up with high frequency using a selection test that is based on the difference in ultraviolet light sensitivity of homoduplex and heteroduplex φX174 RF DNA. Heteroduplex φX174 RF DNA is two to three times more sensitive to ultraviolet light irradiation than is homoduplex φX174 RF DNA (Baas &; Jansz, 1971,1972). Phage DNA derived from single plaque lysates of two of the six mutant complementary strand DNA preparations yielded, after annealing with wild-type complementary strand DNA, heteroduplex DNA with high frequency. DNA sequence analysis in the origin region of RF DNA obtained from these two phage preparations revealed the presence of the expected mutation. RFI DNA of these two origin mutants was nicked by φX174 gene A protein in the same way as wild-type φX174 RFI DNA.Phage DNA derived from single plaque lysates of the other four mutant complementary strand DNA preparations yielded exclusively homoduplex DNA after annealing with wild-type complementary strand DNA. It is concluded that priming with these deoxyhexadecamers resulted in the synthesis of complementary φX174 DNA with lethal mutations. The implications of these results, the construction of two silent, viable φX174 origin mutants and the failure to detect four others, for the initiation mechanism of φX174 RF DNA replication are discussed.     

Structure of RNAs replicated by the DNA-dependent T7 RNA polymerase.

The DNA-dependent RNA polymerase of bacteriophage T7 efficiently and specifically replicates two structurally related RNAs, termed X and Y RNAs. Replication of both RNAs involves synthesis of complementary strands initiated with pppC and pppG. RNAs transcribed from DNA template containing the established sequences of X and Y RNAs were efficiently replicated by T7 RNA polymerase. Both RNAs possess palindromic sequences with a dual axis of symmetry, permitting formation of hairpin-, dumbbell-, or cloverleaf-type structures. The template must consist of RNA and not DNA sequence, and the terminal unpaired dinucleotides of the RNA are necessary for replication. Nucleotidyl transferase activity of E. coli adenylates the unpaired CCOH dinucleotide at the 3' end of a C strand of X RNA. This feature, as well as the length (64 nucleotides) and compact structure of X and Y RNAs, suggests that they may resemble tRNA molecules and tRNA-like structures at the 3' termini of many plant viral RNA genomes.     

Genetic Expression in Heterozygous Replicative Form Molecules of phiX174

Heterozygous replicative form molecules of bacteriophage X174 deoxyribonucleic acid (DNA) have been constructed in vitro. These are composed of viral strands extracted from purified preparations of phage bearing ts mutations and complementary strands of either half length or full length synthesized with purified DNA polymerase, in vitro, on DNA from am3 phage. In infections with such heterozygous DNA, involving mutations in each of four different cistrons, phage with the genotype of the complementary strand comprised 1 to 20% of the total phage produced by a spheroplast population. From single-burst analysis of the progeny from DNA heterozygous in one cistron (B), it appears that those phage with the genotype of the complementary strand arise as major components in a small proportion of the infected cells rather than comprising a minor component in most cells. The implications of such a pattern of expression are discussed with respect to mechanisms of phage DNA synthesis.     

Replication of RNA by the DNA-dependent RNA polymerase of phage T7

The DNA-dependent RNA polymerase of bacteriophage T7 utilizes a specific RNA as a template and replicates it efficiently and accurately. The RNA product (X RNA), approximately 70 nucleotides long, is initiated with either pppC or pppG and contains an AU-tich sequence. Replication of X RNA involves synthesis of complementary strands. Both strands are also significantly self-complementary, producing RNA with an extensive hairpin secondary structure. Replication of X RNA by T7 RNA polymerase is both template and enzyme specific. No other RNA serves as template for replication; neither do other polymerases, including the closely related T3 RNA polymerase, replicate X RNA. The T7 RNA polymerase-X RNA system provides an interesting model for studying replication of RNA by DNA-dependent RNA polymerases. Such a mechanism has been proposed to propagate viroids and hepatitis delta, pathogenic RNAs whose replication seems to depend on cellular RNA polymerases.     

Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. III. A polymerase-primase interaction governs primer size.

Studies with a rolling-circle DNA replication system reconstituted in vitro with a tailed form II DNA template, the DNA polymerase III holoenzyme (Pol III HE), the Escherichia coli single-stranded DNA binding protein, and the primosome, showed that within the context of a replication fork, the oligoribonucleotide primers that were formed were limited to a length in the range of 9 to 14 nucleotides, regardless of whether they were subsequently elongated by the lagging-strand DNA polymerase. This is in contrast to the 8-60-nucleotide-long primers synthesized by the primosome in the absence of DNA replication on a bacteriophage phi X174 DNA template, although when primer synthesis and DNA replication were catalyzed concurrently in this system, the extent of RNA polymerization decreased. As described in this report, we therefore examined the effect of the DNA Pol III HE on the length of primers synthesized by primase in vitro in the absence of DNA replication. When primer synthesis was catalyzed either: i) by the primosome on a phi X174 DNA template, ii) by primase on naked DNA with the aid of the DnaB protein (general priming), or iii) by primase alone at the bacteriophage G4 origin, the presence of the DNA Pol III HE in the reaction mixtures resulted in a universal reduction in the length of the heterogeneous RNA products to a uniform size of approximately 10 nucleotides. dNTPs were not required, and the addition of dGMP, an inhibitor of the 3'----5' exonuclease of the DNA Pol III HE, did not alter the effect; therefore, neither the 5'----3' DNA polymerase activity nor the 3'----5' exonuclease activity of the DNA Pol III HE was involved. E. coli DNA polymerase I, and the DNA polymerases of bacteriophages T4 and T7 could not substitute for the DNA Pol III HE. The Pol III core plays a crucial role in mediating this effect, although other subunits of the DNA Pol III HE are also required. These observations suggest that the association of primase with the DNA Pol III HE during primer synthesis regulates its catalytic activity and that this regulatory interaction occurs independently of, and prior to, formation of a preinitiation complex of the DNA Pol III HE on the primer terminus.     

DNA synthesis in nucleotide-permeable Escherichia coli cells. VII. Conversion of phi chi-174 DNA to its replicative form

On incubation with deoxynucleoside triphosphates and rATP, ether-treated (nucleotide-permeable) cells convert the single-stranded DNA of adsorbed bacteriophage φX174 particles to the double-stranded replicative forms. The main final product is the doubly-closed replicative form, RFI; a minor product is the relaxed form II. Interruptions in the nascent complementary strand of the viral DNA result in pieces corresponding to 5 to 10% of the unit length of the viral DNA. Pieces of similar size were previously seen in studies of the replication synthesis of Escherichia, coli DNA in ether-treated cells. Since the conversion of the single-stranded φX174 DNA to replicative form is known to be mediated entirely by host factors, it is argued that the viral single strands are replicated by macromolecular factors involed in the replication of E. coli DNA and that this is the reason why new φX174 DNA appears in short pieces. Possible consequences of this interpretation for an understanding of duplex replication are discussed. The joining of the short pieces of complementary φX174 DNA is inhibited at low deoxynucleoside triphosphate concentration (1 μM) but not by nicotinamide mononucleotide, which inhibits the NAD-dependent DNA ligase and blocks the conversion of RFII to RFI in ether-treated cells. The results are discussed with respect to previous studies on cell-DNA synthesis (Geider, 1972). It is argued that there are two polynucleotide joining mechanisms, of which only one requires NAD-dependent ligase action.     

A role for single-strand breaks in bacteriophage phi-X174 genetic recombination

Formation of genetic recombinants in bacteriophage φX174 is stimulated up to 50-fold in host cells carrying the recA⁺ allele by subjecting the virus particles to ultraviolet irradiation before infection, or by starving the host cell for thymine during infection; in recA host strains no such increases are observed.φX174 replicative form DNA molecules formed in vivo from ultraviolet-irradiated bacteriophage consist of an intact, circular full-length viral (+) strand and a partially complete complementary (?) strand extending from the point of origin of complementary strand DNA synthesis to an ultraviolet lesion. φX174 replicative form DNA molecules formed in thymine-deficient host strains during thymine starvation have nearly complete circular viral (+) and complementary (?) strands, which contain random single-strand nicks or gaps.Correlation of these structures with the observed increases in recombination suggests that single-strand “breaks” are aggressive intermediate structures in the formation of φX174 genetic recombinants mediated by the host recA⁺ gene product.     

Replication of phi X174 dna with purified enzymes. I. Conversion of viral DNA to a supercoiled, biologically active duplex

Conversion of phi X174 viral, single-stranded circular DNA to the duplex replicative form (RF), previously observed with partially purified enzymes, has now been demonstrated with the participation of 12 nearly pure Escherichia coli proteins containing approximately 30 polypeptides. To complete the synthesis of a full length complementary strand, E. coli DNA polymerase I was needed to fill the short gap left by DNA polymerase III holoenzyme, and to remove the primer and replace it with DNA. Production of supercoiled RF required the further actions of E. coli DNA ligase and gyrase. Net synthesis of viral circles was obtained by coupling the formation of RF supercoils to the actions of the phi X174-encoded gene A protein and E. coli rep protein. Viral DNA circles produced from enzymatically synthesized supercoiled RF, serving as template-substrate, were indistinguishable from those produced from RF isolated from infected cells; synthetic RF and the viral circles generated from it by replication were as biologically active in transfection of spheroplasts as the forms obtained from infected cells and virions. The conversion of single-stranded circular DNA to RF is suggested here as a model for discontinuous synthesis of the lagging strand of the E. coli chromosome. The primosome, a complex of some of the replication proteins responsible for initiations of DNA chains, will be described elsewhere. Multiplication of RF supercoils, described in the succeeding paper, proceeds by a rolling-circle mechanism in which the synthesis of viral strands may have analogies to the continuous synthesis of the leading strand of the E. coli chromosome.     

Structure of an intermediate in the replication of bacteriophage φX174 deoxyribonucleic acid: The initiation site for DNA replication

Replicative form DNA composed of a closed complementary strand and a discontinuous viral strand has been isolated from cells infected with bacteriophage φX174 during the period of single-strand DNA synthesis. This RFII DNA was degraded by the restriction enzyme from Hemophilus influenzae, endonuclease R, and the products analyzed by polyacrylamide gel electrophoresis. The results indicate that there are two types of discontinuity in the viral strands of these molecules: (1) 65% of the molecules contain a gap, which causes a discrete increase in mobility of a specific restriction enzyme fragment, R3. This gap can be selectively repaired with Escherichia coli DNA polymerase I and nucleoside triphosphates, but the molecules are not converted to RFI by addition of E. coli polynueleotide ligase to the reaction mixture. Approximately 30 moles of radioactive TTP are incorporated per mole of RF DNA. (2) 35% of the RF molecules contain a discontinuity, which does not result in a detectable change in mobility of any restriction enzyme fragment. These RF molecules can be converted to RFI by the action of ligase and polymerase I in the presence of nucleoside triphosphates, with incorporation of only approximately one mole of radioactive TTP, specifically into fragment R3, per mole of RF DNA.When the reaction of late RFII DNA and polymerase I is allowed to proceed beyond the repair of the discontinuity, radioactive nucleotides are incorporated into endonuclease R fragments adjacent to R3 in the 5′ → 3′ direction. This technique was utilized to determine a partial order of endonuclease R fragments in φX174.These results suggest that the synthesis of single-strand DNA is initiated from a unique point in cistron A and proceeds clockwise round the φX174 genetic map (cistron order: ABCDEFGH). A comparison of these results with other studies on φX174 suggests that DNA synthesis in all stages of φX174 replication may be initiated from a specific locus on the genome, at or near cistron A.     

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.     

Location of the 5-Methylcytosine Group on the Bacteriophage φX174 Genome

Bacteriophage phiX174 DNA was labeled in vivo with [methyl-(3)H]methionine. The methyl-labeled progeny DNA was extracted from purified bacteriophage phiX174 particles and was used as template for in vitro synthesis of the complementary strand in the presence of the nucleoside triphosphates and Escherichia coli polymerase I. The resultant replicative form DNA was then cleaved, in separate experiments, with restriction endonucleases from Haemophilus influenzae and H. aegyptius. The DNA fragments were analyzed by polyacrylamide gel electrophoresis. It is concluded that the single methylcytosine in the viral DNA is located in a specific region of the phiX174 genome, very likely in gene H.     

Template-switching during DNA synthesis by Thermus aquaticus DNA polymerase I.

Recombinant DNA molecules are often generated during the polymerase chain reaction (PCR) when partially homologous templates are available [e.g., see Pääbo et al. (1990) J. Biol. Chem. 265, 4718-4721]. It has been suggested that these recombinant molecules are a consequence of truncated extension products annealing to partially homologous templates on subsequent PCR cycles. However, we demonstrate here that recombinants can be generated during a single round of primer extension in the absence of subsequent heat denaturation, indicating that template-switching produces some of these recombinant molecules. Two types of template-switches were observed: (i) switches to pre-existing templates and (ii) switches to the complementary nascent strand. Recombination is reduced several fold when the complementary template strands are physically separated by attachment to streptavidin magnetic beads. This result supports the hypothesis that either the polymerase or at least one of the two extending strands switches templates during DNA synthesis and that interaction between the complementary template strands is necessary for efficient template-switching.     

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.     

The role of palindromic and non-palindromic sequences in arresting DNA synthesis in vitro and in vivo

The nature of specific DNA sequences that arrest synthesis by mammalian DNA polymerase alpha in vitro was analyzed using circular, single-stranded M13 or phi X174 virion DNA templates annealed to a unique, terminally labeled, DNA primer. This method rigorously defined both the starting nucleotide position and the direction of synthesis, as well as making the amount of radioactivity proportional to the number rather than the length of nascent DNA chains. The precise nucleotide locations of arrest sites were determined over templates with complementary sequences by cloning unique DNA restriction fragments into M13 DNA and isolating virions containing either the Watson or Crick strand. Results were correlated with the locations of palindromic (self-complementary) sequences, repeated sequences, and repeated sequences with mirror-image orientation. Two classes of DNA synthesis arrest sites were identified, distinct in structure but equivalent in activity. Class I sites consisted of palindromic sequences that formed a stable hairpin structure in solution and arrested DNA polymerase on both complementary templates. The polymerase stopped precisely at the base of the duplex DNA stem, regardless of the direction from which the enzyme approached. Class II sites consisted of non-palindromic sequences that could not be explained by either secondary structure or sequence symmetry elements, and whose complementary sequence was not an arrest site. Size limits, orientation and some sequence specificity for arrest sites were suggested by the data. Arrest sites were also observed in vivo by mapping the locations of 3'-end-labeled nascent simian virus 40 DNA strands throughout the genome. Arrest sites closest to the region where termination of replication occurs were most pronounced, and the locations of 80% of the most prominent sites appeared to be recognized by alpha-polymerase on the same template in vitro. However, class I sites were not identified in vivo, suggesting that palindromic sequences do not form hairpin structures at replication forks.     

DNA crosslinks,single-strand breaks and effects on bacteriophage T4 survival from tritium decay of [2-3H]adenine, [8-3H]adenine and [8-3H]guanine

Escherichia coli and bacteriophage T4 DNA containing [2-³H]adenine accumulated crosslinks between the complementary strands. For T4 DNA stored in frozen solution there were 0.41 to 0.54 crosslinks formed per tritium decay. The crosslinks were demonstrated both by an increased DNA sedimentation rate in alkaline sucrose gradients and by an increasing amount of DNA that renatured quickly after denaturation by heat or alkali. Single-strand breaks were also formed with an efficiency of 0.08 to 0.50 breaks per tritium decay. DNA containing both [8-³H]adenine and [8-³H]guanine showed no crosslinking but did undergo single-strand breaks at a rate of 0.08 per tritium decay. T4 bacteriophage containing [2-³H]adenine lost plaque-forming ability when stored at 4 °C, with 0.34 lethal hits per tritium decay, whereas the same phage labeled with a mixture of [8-³H]adenine and [8-³H]guanine sustained only 0.12 lethal hits per tritium decay. The loss of plaque-forming ability in the latter case is probably due to a radiation effect from the emitted beta particle; the high lethal efficiency for tritium decay at 2-adenine is probably caused either by crosslinks between complementary strands or from some undetected lesion produced in the DNA.     

The A* protein of phi X174 is an inhibitor of DNA replication

Extracts prepared from phi X174 infected E. coli cells inhibited in vitro RF replication The inhibition was dependent upon the presence of A* protein in the reaction and served as an assay to highly purify the A* protein. Purified A* protein bound tightly to duplex DNA as well as single-stranded DNA. The binding of the A* protein to duplex DNA inhibited (I) its single-stranded DNA specific endonucleolytic activity; (II) in vitro synthesis of viral (+) single stranded DNA on an A-RFII DNA complex template; (III) ATP hydrolysis by rep protein and unwinding of the strands of RF DNA. We propose that this inhibitory activity is responsible in vivo for the shut off of E. coli chromosome replication during phi X174 infection, and has a role in the transition from semiconservative RF DNA replication to single-stranded DNA synthesis in the life cycle of phi X174.     

Fine mapping of secondary structures of fd phage DNA in the region of the replication origin.

A synthetic heptaribonucleotide, GACCCCC, which is complementary to a unique site on fd bacteriophage DNA, primes DNA synthesis of fd by T4 bacteriophage DNA polymerase. The rate of the GACCCCC-primed DNA synthesis was not uniform as reflected by the appearance of discrete DNA fragments as replication intermediates on an alkaline agarose gel. After 10 minutes of synthesis a significant fraction of the DNA product ran as a single band with a length of about 1960 nucleotides. We have isolated this DNA fragment, hybridized back to unlabeled fd DNA template, and mapped the Taq I restriction fragments by urea polyacrylamide gel electrophoresis. This fine mapping procedure has located two major pause sites at fd nucleotide positions 5575 and 5674. These sites reside in the stem of two very stable hairpin helices near the origin of DNA replication of fd. Models for the functional roles of these two hairpin helices are presented.     

Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication

The results presented in this paper indicate that the phi 29 DNA polymerase is the only enzyme required for efficient synthesis of full length phi 29 DNA with the phi 29 terminal protein, the initiation primer, as the only additional protein requirement. Analysis of phi 29 DNA polymerase activity in various in vitro DNA replication systems indicates that two main reasons are responsible for the efficiency of this minimal system: 1) the phi 29 DNA polymerase is highly processive in the absence of any accessory protein; 2) the polymerase itself is able to produce strand displacement coupled to the polymerization process. Using primed M13 DNA as template, the phi 29 DNA polymerase is able to synthesize DNA chains greater than 70 kilobase pairs. Furthermore, conditions that increase the stability of secondary structure in the template do not affect the processivity and strand displacement ability of the enzyme. Thus, the catalytic properties of the phi 29 DNA polymerase are appropriate for a phi 29 DNA replication mechanism involving two replication origins, strand displacement and continuous synthesis of both strands. The enzymology of phi 29 DNA replication would support a symmetrical model of DNA replication.     

Conditional coupling of leading-strand and lagging-strand DNA synthesis at bacteriophage T4 replication forks

Eight proteins encoded by bacteriophage T4 are required for the replicative synthesis of the leading and lagging strands of T4 DNA. We show here that active T4 replication forks, which catalyze the coordinated synthesis of leading and lagging strands, remain stable in the face of dilution provided that the gp44/62 clamp loader, the gp45 sliding clamp, and the gp32 ssDNA-binding protein are present at sufficient levels after dilution. If any of these accessory proteins is omitted from the dilution mixture, uncoordinated DNA synthesis occurs, and/or large Okazaki fragments are formed. Thus, the accessory proteins must be recruited from solution for each round of initiation of lagging-strand synthesis. A modified bacteriophage T7 DNA polymerase (Sequenase) can replace the T4 DNA polymerase for leading-strand synthesis but not for well coordinated lagging-strand synthesis. Although T4 DNA polymerase has been reported to self-associate, gel-exclusion chromatography displays it as a monomer in solution in the absence of DNA. It forms no stable holoenzyme complex in solution with the accessory proteins or with the gp41-gp61 helicase-primase. Instead, template DNA is required for the assembly of the T4 replication complex, which then catalyzes coordinated synthesis of leading and lagging strands in a conditionally coupled manner.