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.     

The mechanism of replication of phi X174 single-stranded DNA. II. The role of viral proteins

We have studied the replication of φX174 DNA in Escherichia coli infected with various amber mutants (cistrons I to VII) of φX. Previous research showing that some of these mutants are able to form replicative form (RF) DNA but are unable to produce net amounts of viral progeny single-stranded DNA has been confirmed and extended. Evidence is presented that a defect in any one of four viral cistrons prevents the asymmetric replication of the RF to produce progeny viral DNA. At least four virus-coded proteins, three of which are part of the mature virion, must be present before single-stranded DNA synthesis can even be initiated; the possibility that single-stranded DNA is made and then degraded or converted to RF is eliminated. Mutants in one cistron (II) do permit the asymmetric replication of RF at late times, but the displaced viral strand is incorporated into a defective particle and subsequently may be partially degraded. Both RFI (superhelix) and RFII are present in roughly comparable amounts throughout the normal latent period in infections with wild-type phage or any of the phage mutants.     

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.     

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.     

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.     

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.     

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.     

Bacteriophage phi X174 RF DNA replication in vivo. A study by electron microscopy.

We have investigated bacteriophage φX174 RF ² DNA replication by electron microscopy. Three different, types of replicative intermediates were observed: rolling circles, partially duplex DNA circles and structures consisting of two DNA circles connected at a single point.Rolling circles with a single-stranded or partially double-stranded DNA tail were both observed. After cleavage of the rolling circles with the restriction endonuclease from Providentia stuartii 164 (PstI) the startpoint of rolling circle replication could be located at 21 map units from the PstI cleavage site in agreement with the previously determined position of the origin of φX RF DNA replication.Partially duplex DNA circles consist of circular viral DNA strands and incomplete complementary DNA strands. After cleavage of these molecules with PstI information about the startpoints of the synthesis of the complementary DNA strand was obtained.The connected DNA circles always contain one completely double-stranded DNA circle whereas the other circle consists of either single-stranded, partially duplex or completely duplex DNA.Part of the duplex-to-duplex DNA circles represent the well-known figure eight or catenated circular dimers. The other connected DNA circles presumably represent replication intermediates which arise by the association of the end of the genome length tail of the rolling circle with the origin-terminus region. This is suggested by the fact that the point of contact between the two DNA circles is located at approximately 21 map units from the Pst1 cleavage site, i.e. at the origin-terminus region of the φX genome. The connected DNA circles may be intermediates in the circularization and cleavage of the genome-length tail of the rolling circles in vivo.A model for φX174 RF DNA replication in vivo summarizing the data obtained by biochemical (Baas et al., 1978) and electron microscopic analysis of replicative intermediates is presented (Fig. 9).     

A rolling circle model of the in vivo replication of bacteriophage phiX174 replicative form DNA: different fate of two types of progeny replicative form

In a preceding paper (Schröder and Kaerner, 1972) a rolling circle mechanism has been described for the replication of bacteriophage φX174 replicative form. Replication involved nicking and elongation of the viral (positive) strand component of the RF molecule resulting in the displacement of a single-strand tail of increasing length. The synthesis of the new complementary (negative) strand on the single-strand tails appears to be initiated with considerable delay and converts the tail into double-stranded DNA. Before the new negative strand is completed the replicative intermediates split into (I) a complete RF molecule containing the “old” negative and the new positive strand, and (II) a linear, partially double-stranded “tail” consisting of the complete old positive strand and a fragment of the new negative strand.The present study is concerned with the fate during RF replication of these fragments of the rolling circles. Those RFII molecules containing the old negative strands appear to go into further replication rounds repeatedly. Some of the tails were found in the infected cells in their original linear form. “Gapped” RFII molecules, which have been described earlier by Schekman and co-workers (Schekman &; Ray, 1971; Schekman et al., 1971), are supposed to originate from the tails of rolling circle intermediates by circularization of their positive strand components. Evidence is provided by our experiments that even late during RF replication these gaps are present only in the negative strands of RFII. Appropriate chase experiments indicated that the tails finally are converted to RFI molecules. Progeny RFI molecules could not be observed to start new replication rounds under our conditions although we cannot exclude that this might happen to some minor extent.The results presented suggest that the master templates for RF replication are the first negative strands to be formed, rather than the parental positive strands.     

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.     

Lack of repair of ultraviolet light damage in Mycoplasma gallisepticum

Molecules with single-stranded tails (rolling circles) were isolated as replicating intermediates in G4 progeny single-stranded DNA synthesis. Lysates from infected cells harvested late in infection during single-stranded DNA synthesis were not deproteinised but analysed directly in caesium chloride and propidium diiodide gradients. The gradient fractionated them on the basis of tail length. If the lysates were first deproteinised however, the tailed replicative intermediates banded as a peak at a density just greater than that of replicative form II DNA (RFII) and did not spread down the gradient. The origin of synthesis of the viral strand tail was mapped by electron microscopy as 55 to 60% away from the single EcoRI cleavage site. Termination molecules finishing a round of viral strand DNA synthesis have been identified as molecules consisting of a closed single-stranded DNA circle attached by a very small region to the parent double-stranded DNA circle.     

The process of infection with bacteriophage φX174: XXXIX. The structure of a DNA form with restricted binding of intercalating dyes observed during synthesis of φX single-stranded DNA

A DNA form with restricted binding of intercalating dyes (propidium iodide or ethidium bromide) has been found in bacteriophage φX-infected cells during the period of single-stranded DNA synthesis. In the electron microscope, this DNA form is seen to be a double-stranded DNA ring with two single-stranded DNA tails protruding from the same portion of the ring; it is composed of a linear φX DNA strand, longer than one φX genome, and a single-stranded ring complementary to φX DNA. Base-pairing of these two tails in partially complementary regions restricts unwinding of the double-stranded DNA ring and consequently intercalation and binding of the dyes. It is postulated that these molecules originate from a previously reported precursor of φX DNA, namely a double-stranded ring with a single-stranded tail, by branch migration.     

Isolation of specific ribosome binding sites from single-stranded DNA.

The ability of Escherichia coli ribosomes to protect small specific regions of single-stranded bacteriophage DNA from digestion by pancreatic DNAase has been investigated. A procedure is described by which ribosome-protected fragments can be isolated from the DNA of bacteriophage f1 and φX174. Size determination by polyacrylamide gel electrophoresis or thin layer homochromatography together with fingerprinting analysis following chemical depurination or digestion with E. coli endonuclease IV were employed to show that these fragments represent a small specific portion of these DNAs. The protection reaction is largely dependent upon components necessary for ribosome binding to mRNA, including GTP, formylmethionyl-tRNA, and initiation factors. Thus, ribosomal binding to DNA mimics the ribosome-mRNA interaction. Furthermore, the regions in f1 and φX174 DNA which are protected differ in sequence from each other.When E. coli endonuclease IV is substituted for pancreatic DNAase in the ribosome protection reaction, a fragment of φX174 DNA is obtained about 150 bases in length which contains all of the pyrimidine tracts in the shorter 50-base fragment obtained with pancreatic DNAase, and a number of additional polypyrimidines.Double-stranded DNAs such as φX174 replicative form do not bind at all to ribosomes in their native state. Heat denaturation of such double-stranded DNAs allows ribosome binding. Protection of the same specific regions as those protected in single-stranded φX174 DNA was observed. A similar specific protection was observed following heat denaturation and ribosome binding with DNA from polyoma virus.     

Identification of a phi X174 coded protein involved in the inhibition of beta-galactosidase synthesis in Escherichia coli

The synthesis of β-galactosidase (EC 3.2.1.23: β-D-galactoside galactohydrolase) in $\text{Escherichia}\text{coli}$ is repressed as a result of infection with single-stranded DNA phage ØX174. An $\text{amber}$ mutant in ØX174 cistron A, which codes for two proteins, does not inhibit the enzyme synthesis while $\text{amber}$ mutants in all other genes do cause repression. A mutant near the amino-terminal end of cistron A, which produces the small 35,000 molecular weight cistron A polypeptide, also inhibits the synthesis of β-galactosidase. Inhibition is also observed in an $\text{Escherichia}\text{coli}\text{rep}$ mutant which does not support the replication of replicative-form DNA. Exogenous nucleotide bases and cyclic 3′,5′-adenosine monophosphate (cyclic AMP) do not have any effect on the degree of repression.     

Formation of the parental replicative form DNA of bacteriophage phi-X174 and initial events in its replication

Intracellular φX174 DNA was studied under a variety of conditions that prevent the replication of the parental replicative form DNA. These conditions included treatment with 150 μg of chloramphenicol per ml., the use of the rep₃⁻ mutation of the host cell, amber mutation (am 8) in the viral gene responsible for RF replication (gene A) ^† and combinations thereof. In all cases the majority of the parental RF was in the covalently closed form (RFI). The relative amount of RF with a discontinuity in one strand (RFII) in these cases was between 2 and 10% of the total RF and independent of the multiplicity of infection. The only exception was seen in infections of rep₃ cells with φX am 3 (a mutant in the lysis gene, gene E, used as a wild-type representative). In this case a fairly constant absolute amount of RFII (1 to 4 per cell), independent of the multiplicity of infection, was formed, consisting almost exclusively of a closed complementary and an open parental viral strand. Since the formation of this type of RFII was dependent on protein synthesis and the presence of the product of φX gene A, it is concluded that the discontinuity in the parental viral strand represents the result of the action of the gene A product on the DNA. Possible mechanisms for the mode of action of the gene A product are discussed.     

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.     

In vivo methylation of replicating bacteriophage phi chi174 DNA

The pattern of DNA methylation during the infection of Escherichia coli C cells with bacteriophage φX174, has been studied. In vivo methylated DNA was isolated and analyzed using the following techniques: velocity sedimentation through neutral and alkaline sucrose gradients, isopycnic analysis, chromatography on benzoylated DBAE-cellulose columns and specific enzymatic digestion. All these analytical methods indicated that the DNA molecules that are methylated during the process of phage φX DNA replication are the replicating intermediates composed of a circular complementary strand and a viral strand larger than one genome length. It is concluded that methylation occurs on the nascent DNA strand of the replicating intermediates involved in the synthesis of progeny single-stranded DNA.