Rolling-circle replication of UV-irradiated duplex DNA in the phi X174 replicative-form----single-strand replication system in vitro.

Cloning of the phi X174 viral origin of replication into phage M13mp8 produced an M13-phi X174 chimera, the DNA of which directed efficient replicative-form----single-strand rolling-circle replication in vitro. This replication assay was performed with purified phi X174-encoded gene A protein, Escherichia coli rep helicase, single-stranded DNA-binding protein, and DNA polymerase III holoenzyme. The nicking of replicative-form I (RFI) DNA by gene A protein was essentially unaffected by the presence of UV lesions in the DNA. However, unwinding of UV-irradiated DNA by the rep helicase was inhibited twofold as compared with unwinding of the unirradiated substrate. UV irradiation of the substrate DNA caused a strong inhibition in its ability to direct DNA synthesis. However, even DNA preparations that contained as many as 10 photodimers per molecule still supported the synthesis of progeny full-length single-stranded DNA. The appearance of full-length radiolabeled products implied at least two full rounds of replication, since the first round released the unlabeled plus viral strand of the duplex DNA. Pretreatment of the UV-irradiated DNA substrate with purified pyrimidine dimer endonuclease from Micrococcus luteus, which converted photodimer-containing supercoiled RFI DNA into relaxed, nicked RFII DNA and thus prevented its replication, reduced DNA synthesis by 70%. Analysis of radiolabeled replication products by agarose gel electrophoresis followed by autoradiography revealed that this decrease was due to a reduction in the synthesis of progeny full-length single-stranded DNA. This implies that 70 to 80% of the full-length DNA products produced in this system were synthesized on molecules that carried photodimers. Thus, similarly to its activity on UV-irradiated single-stranded DNA, DNA polymerase III holenzyme can bypass pyrimidine photodimers in the more complex replicative form --->single-strand replication, which involves, in addition to the polymerizing activity, the unwinding of the duplex by the rep helicase and the participation of a more complex multiprotein replisome.     

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.     

Effect of phi X C protein on leading strand DNA synthesis in the phi X174 replication pathway

The influence of the bacteriophage phi X174 (phi X) C protein on the replication of bacteriophage phi X174 DNA has been examined. This small viral protein, which is required for the packaging of phi X DNA into proheads, inhibits leading strand DNA synthesis. The inhibitory effect of the phi X C protein requires a DNA template bearing an intact 30-base pair (bp) phi X origin of DNA replication that is the target site recognized by the phi X A protein. Removal of nucleotides from the 3' end of this 30-bp conserved origin sequence prevents the inhibitory effects of the phi X C protein. Leading strand replication of supercoiled DNA substrates containing the wild-type phi X replication origin results in the production of single-stranded circular DNA as well as the formation of small amounts of multimeric and sigma structures. These aberrant products are formed when the termination and reinitiation steps of the replication pathway reactions are skipped as the replication fork moves through the origin sequence. Replication carried out in the presence of the phi X C protein leads to a marked decrease in these aberrant structures. While the exact mechanism of action of the phi X C protein is not clear, the results presented here suggest that the phi X C protein slows the movement of the replication fork through the 30-bp origin sequence, thereby increasing the fidelity of the termination and reinitiation reactions. In keeping with the requirement for the phi X C protein for efficient packaging of progeny phi X DNA into proheads, the phi X C protein-mediated inhibition of leading strand synthesis is reversed by the addition of proteins essential for phi X bacteriophage formation. Incubation of plasmid DNA substrates bearing mutant 30 base pair phi X origin sequences in the complete packaging system results in the in vitro packaging and production of infectious particles in a manner consistent with the replication activity of the origin under study.     

Purification and properties of Escherichia coli protein i, a prepriming protein in phi X174 DNA replication

Protein i, one of seven Escherichia coli proteins essential for primosome initiation of DNA chains in the in vitro conversion of single-stranded phi X174 DNA to duplex replicative form, has been purified approximately 15,000-fold to more than 98% purity. The protein is an oligomer of 22,000-dalton subunits migrating as a single electrophoretic band on native, as well as on denaturing polyacrylamide gels. Estimates of a Stokes radius of 41 A, a sedimentation coefficient of 3.5 S, a Mr = 61,000, and a frictional coefficient of 1.57 suggest that native protein i is a highly asymmetric oligomer composed of three identical subunits. About 50 such molecules are present/cell. Cross-linking the protein with dimethylsuberimidate or dimethyladipimidate produced three major bands corresponding to the monomer, dimer, and trimer, as well as two minor bands corresponding to the tetramer and pentamer. Incorporation of 3H-labeled "trimeric" protein i into the prepriming replication intermediate (primosome) occurs at a stage requiring participation of dnaB and dnaC proteins, and follows the actions of proteins n, n', and n". After extension of primers by DNA polymerase III holoenzyme, protein i is not retained in the isolated primosome complex. Thus, protein i is essential in the assembly of a functional primosome, but its precise physiologic role and genetic locus are still unknown.     

Enzymatic properties of the bacteriophage phi X174 A protein on superhelical phi X174 DNA: a model for the termination of the rolling circle DNA replication.

Incubation of phi X174 replication form I DNA with the A* protein of phi X174 in the presence of MN2+ results in the formation of three different types of DNA molecules: open circular form DNA (RFII), linear form DNA (RFIII) and the relaxed covalently closed form DNA (RFIV). The RFII and RFIII DNAs are shown to be A* protein-DNA complexes by electron microscopy using the protein labeling technique of Wu and Davidson (1). The linear double-stranded RFIII DNA molecule carries at one end a covalently attached A* protein whereas at the other end of the molecule the single-stranded termini are covalently linked to each other. The structure of the RFIII DNA shows its way of formation. The described properties of the A* protein indicate the way the larger A protein functions in the termination step of the rolling-circle type of phi X174 DNA replication.     

The mechanism of replication of phi X 174. XVIII. Gene A and A* proteins of phi X 174 bind tightly to phi X 174 replicative form DNA

Evidence is presented that the gene A and A * proteins of bacteriophage phi X 174 form covalent associations with the 5' ends of the DNA molecules when superhelical phi X replicative form DNA is nicked by a combination of these proteins in vitro. This evidence is: 1, The 5' ends of the DNA molecules nicked by the gene A protein and reacted with bacterial alkaline phosphatase were protected against subsequent phosphorylation by polynucleotide kinase even after treatment of the nicked DNA with SDS and pronase followed by centrifugation on a high-salt neutral sucrose gradient. 2, Iodinated pronase-sensitive material remained attached to the nicked replicative form DNA and could not be removed by exposure to SDS or 2 M NaCl, either by sedimentation through high-salt neutral sucrose gradients, or by CsCl equilibrium centrifugation. 3, Iodinated pronase-sensitive material was detected on DNA that had been nicked during the reaction, but not on unreacted DNA. 4, Electrophoresis of the iodinated pronase-sensitive, DNA-bound material in SDS-polyacrylamide gels after DNAse digestion revealed that it was composed almost entirely polypeptides with electrophoretic mobilities similar to those of the gene A and A * proteins. We speculate that the gene * protein may be essential for normal progeny single-stranded DNA synthesis in vivo.     

Formation of rolling-circle molecules during phi X174 complementary strand DNA replication

The primosome is a mobile multiprotein priming apparatus that requires seven Escherichia coli proteins for assembly (the products of the dnaB, dnaC and dnaG genes; replication factor Y (protein n'); and proteins i, n, and n"). While the primosome is analagous to the phage T7 gene 4 protein and phage T4 gene 41/61 proteins in its DNA G-catalyzed priming function, its ability to act similarly also as a DNA helicase has remained equivocal. The role of the primosome in unwinding duplex DNA strands was investigated in the coliphage phi X174 SS(c)----replicative form DNA replication reaction in vitro, which requires the E. coli single-stranded DNA binding protein, the primosomal proteins, and the DNA polymerase III holoenzyme. Multigenome-length, linear, double-stranded DNA molecules were generated in this reaction, presumably via a rolling circle-type mechanism. Synthesis of these products required the presence of a helicase-catalyzed strand-displacement activity to permit multiple cycles of continuous complementary (-) strand synthesis. The participation of the primosome in this helicase activity was supported by demonstrating that other SS(c) DNA templates (G4 and alpha-3), which lack primosome assembly sites, failed to support significant linear multimer production and that replication of phi X174 with the general priming system (the DNA B and DNA G proteins and DNA polymerase III holoenzyme) resulted in a 13-fold lower rate of linear multimer synthesis.     

Fidelity of replication of bacteriophage phi X174 DNA in vitro and in vivo

Seven different revertants of bacteriophage phi X174am16 (AB5276G leads to T) have been isolated and the nature of the reversions determined by sequencing their DNA. The revertants each differ from am16 by just a single base substitution. These may be distinguished with varying degrees of ease by characteristic temperature sensitivities of growth. This has facilitated the determination of the frequency at which DNA polymerase III catalyses different types of substitution mutations in copying phi X174 DNA in vitro and in vivo. During the replicative form (RF) leads to single-stranded (SS) stage of replication in vitro, four different revertants may be readily produced according to well-defined rate laws on biasing the concentrations of dNTPs. Transversion mutations are found to be formed predominantly by purine x purine mismatching, whilst transitions are formed predominantly by G x T mismatching. The substitutions via G x T and G x A mismatches are estimated to occur at similar frequencies in vivo. The two most common revertants isolated in vivo, however, are not those readily produced during the RF leads to SS stage in vitro but are those produced on purine x purine mismatching in the SS leads to RF stage. The accuracy of the DNA polymerase in vitro appears to be similar to that in this stage in vivo. However, the overall accuracy of the RF leads to SS replication in vivo is more accurate than predicted from the measurements of the accuracy in vitro.     

Mechanism of replication of bacteriophage phi X174. XXII. Site-specific mutagenesis of the A* gene reveals that A* protein is not essential for phi X174 DNA replication

The A and A* proteins of phage phi X174 are encoded in the same reading frame in the viral genome; the smaller A protein is the result of a translational start signal with the A gene. To differentiate their respective functions, oligonucleotide-directed site-specific mutagenesis was used to change the ATG start codon of the phi X 174 A* gene, previously cloned into pCQV2 under lambda repressor control, into a TAG stop codon. The altered A gene was then inserted back into phi X replicative form DNA to produce an amber mutant, phi XamA*. Two different Escherichia coli amber suppressor strains infected with this mutant produced viable progeny phage with only a slight reduction in yield. In Su+ cells infected with phi XamA*, phi X gene A protein, altered at one amino acid, was synthesized at normal levels; A* protein was not detectable. These observations indicate that the A* protein increases the replicative efficiency of the phage, perhaps by shutting down host DNA replication, but is not required for replication of phi X174 DNA or the packaging of the viral strand under the conditions tested.     

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.     

Initiation and termination of the bacteriophage phi X174 rolling circle DNA replication in vivo: packaging of plasmid single-stranded DNA into bacteriophage phi X174 coats.

The bacteriophage phi X174 viral (+) origin when inserted in a plasmid can interact in vivo with the A protein produced by infecting phi X174 phages. A consequence of this interaction is packaging of single-stranded plasmid DNA into preformed phage coats resulting in infective particles (1). This property was used to study morphogenesis and to analyse the signals for initiation and termination of the rolling circle DNA replication in vivo. It is shown that the size of the DNA had a strong effect on the encapsidation by the phage coats and the infectivity of the particle. Termination was analysed by using plasmids with two phi X (+) origins either in the same orientation or in opposite orientation. Both origins were used with equal frequency. Initiation at one origin resulted in very efficient termination (greater than 96%) at the second origin in the case of two origins in the same orientation. When the two (+) origins have opposite orientations, no correct termination was observed. The second origin in the opposite strand effectively inhibits (greater than 98%) the normal DNA synthesis; i.e. the covalently bound A protein present in the replication fork interacts with the (+) origin sequence in the opposite strand.     

Studies on the role of the phi X174 gene A protein in phi X174 viral strand synthesis. III. Replication of DNA containing two viral replication origins

Supercoiled plasmid bearing two wild-type phi X origin sequences on the same strand supported the phi X A protein-dependent in vitro formation of two smaller single-stranded circles, the lengths of which were equivalent to the distance between the two origins. Additional double origin plasmids were utilized to determine whether origins defective in the initial nicking event (initiation) could support circularization (termination). In all cases tested, the presence of a mutant origin on the same strand with a wild-type origin affected the level of replication in a manner consistent with the previously determined activity of the mutant origin. When a functional mutant origin was present on the same strand as a wild-type origin, the efficiency of replication and the DNA products formed were almost identical to those of the plasmid containing two wild-type origins. Plasmid DNA bearing both a wild-type origin and a mutant origin that did not support phi X A protein binding or nicking activity, on the other hand, supported efficient DNA synthesis of only full-length circular products, indicating that the origin defective for initiation was incapable of supporting termination. In contrast, the presence of a wild-type origin and an origin that did bind the phi X A protein but was not cleaved resulted in a marked decrease in DNA synthesis along with the production of only full-length products. This suggests that the phi X A protein stalls when it encounters a sequence to which it can bind but cannot cleave. Replication of double origin plasmids containing one functional phi X origin on each strand of the supercoiled DNA was also examined. With such templates, synthesis from the wild-type origin predominated, indicating preferential cleavage of the intact origin sequence. Replication of such substrates also produced a number of aberrant structures, the properties of which suggested that interstrand exchange of the phi X A protein had occurred.     

phi X174-directed DNA and protein syntheses in infected minicells.

Phi X174-infected minicells, produced by Escherichia coli PC2251, synthesized 11 phi X174-encoded polypeptides. The infecting single-stranded viral genome was converted to a double-stranded, closed circular, replicative form (replicative form I). Little, if any, replicative form I replication took place, and synthesis of progeny single-stranded molecules could not be detected.     

Mutational analysis of the bacteriophage phi X174 replication origin

Bacteriophage phi X174 mutants within the 30 base-pair replication origin were constructed using oligodeoxynucleotide-directed mutagenesis. A total of 18 viable base substitution mutants at 13 different positions within the origin region were obtained. The majority of these ori mutants have a plaque morphology and burst size comparable to that of wild-type phi X174. Two phi X174 ori mutants with a reduced growth ability spontaneously acquired additional mutations that enhanced the growth rate. The additional mutation was located at the same site as the original mutation or was located in the N-terminal part of the gene A protein. This latter secondary mutation is responsible for a better binding and/or recognition of the gene A protein to the mutated origin. In a Darwinian experiment wild-type phi X174 outgrows all phi X174 ori mutants, indicating the superiority of the wild-type ori sequence for the reproduction of bacteriophage phi 174. Insertions and deletions were constructed at different positions within the phi X174 replication origin cloned in a plasmid. Small insertions and deletions in the A + T-rich spacer region do not inhibit phi X174 gene A protein cleavage in vitro, but severely impair packaging of single-stranded plasmid DNA in viral coats.     

Gene A protein cleavage of recombinant plasmids containing the phi X174 replication origin

Synthetic oligonucleotides, DNA ligase and DNA polymerase were used to construct double-stranded DNA fragments homologous to the first 25, 27 or 30 b.p. of the origin of replication of bacteriophage phi X174 (nucleotides 4299-4328 of the phi X174 DNA sequence). The double-stranded DNA fragments were cloned into the unique SmaI or HindIII restriction sites in the kanamycin-resistance gene of pACYC177 (AmpR, KmR). Recombinant plasmids were picked up by colony hybridization. DNA sequencing showed that not only recombinant plasmids with the expected insert were formed, but also recombinant plasmids with a shorter insert. Recombinant plasmids with an insert homologous to the first 24, 25, 26, 27, 28 or all 30 b.p. of the phi X174 origin region were thus obtained. Supercoiled plasmids containing a sequence homologous to the first 27, 28 or 30 b.p. of the phi X174 origin region are nicked by the phi X174 gene A protein. However, the other supercoiled plasmids are not nicked by the phi X174 gene A protein. These results show that the first 27 b.p. of the phi X174 origin region are sufficient as well as required for the initiation step in phi X174 RF DNA replication, i.e. the cleavage by gene A protein.     

Mechanism of replication of bacteriophage phi X174 XIX. Initiation of phi X174 viral strand DNA synthesis at internal sites on the genome.

Bacteriophage phi X174 viral strand DNA molecules shorter than genome length found late in the infectious cycle in Escherichia coli were 5' end labeled with 32P. Hybridization of the 32P-labeled molecules to restriction enzyme fragments of phi X replicative form DNA revealed an excess of phi X molecules whose 5' ends mapped in HaeIII fragments Z3 and Z4 in comparison with fragments Z1 and Z2. This suggests that initiation of phi X174 viral strand DNA synthesis may occur at internal sites on the complementary strand. There are several appropriately located sequences that might serve as n' (factor Y) recognition sequences and thereby facilitate discontinuous synthesis of the viral strand.     

Effects of palindromes on in vivo DNA replication and mutagenesis in bacteriophage phi X174 RF DNA.

Bacteriophage phi X174 mutants with insertions of palindromic DNA sequences are rapidly outgrown by competing wild-type phage (Müller & Turnage, J. Mol. Biol. 189: 285). The basis for this defect was investigated and found to be due to an exclusion event early in the infectious cycle, in which phage genomes with palindromic inserts were preferentially excluded by wt phage. In addition, we have obtained further evidence for a palindrome induced genetic instability. Both defects are dependent on palindrome size and sequence, consistent with a model which involves formation of cruciforms, or cruciform-like structures. We propose that formation of unusual DNA secondary structures reduces the effectiveness of replicative form (RF) DNA to interact with limiting replication factors or membrane binding sites, possibly because of interaction with the host recombination system.     

Synthesis of bacteriophage phi X174 in vitro: mechanism of switch from DNA replication to DNA packaging

Replication of a replicative form DNA of bacteriophage phi X174 initiates by rolling-circle synthesis of the viral DNA followed by discontinuous synthesis of the complementary DNA. Gene C protein of phi X174, which is involved in DNA packaging, inhibits the rolling-circle DNA synthesis by binding to the initiation complex in vitro. The gene C protein-associated initiation complex can synthesize and package the viral DNA to produce infectious phage when supplemented with phi X174 gene J protein and the prohead. Multiple rounds of phage synthesis occur without dissociation of the gene C protein from the complex. These results indicate that gene C protein is central in the switch from replication of a replicative form DNA to synthesis and concomitant packaging of viral DNA into phage capsid, which occurs in the late stage of infection.     

The role of gene A protein and E. coli rep protein in the replication of phi X 174 replicative form DNA

The gene A protein of bacteriophage phi X 174 initiates replication of super-twisted RFI DNA by cleaving the viral (+) strand at the origin of replication and binding to the 5' end. Upon addition of E. coli rep protein (single-stranded DNA dependent ATPase), E. coli single-stranded DNA binding protein and ATP, complete unwinding of the two strands occurs. Electron microscopic analyses of intermediates in the reaction reveal that the unwinding occurs by movement of the 5' end into the duplex, displacing the viral strand in the form of a single-stranded loop. Since unwinding will not occur in the absence of either gene A protein or rep protein, it is presumed that the rep protein interacts to form a complex with the bound gene A protein. Single-stranded DNA binding protein facilitates the unwinding by binding to the exposed single-stranded DNA. Further addition of the four deoxyribotriphosphates and DNA polymerase III holoenzyme to the reaction results in synthesis of viral (+) single-stranded circles in amounts exceeding that of the input template. A model describing the role of gene A protein and rep protein in duplex DNA replication is presented and other properties of gene A protein discussed.