Orientation of the DNA in the filamentous bacteriophage f1

The filamentous bacteriophage f1 consists of a molecule of circular single-stranded DNA coated along its length by about 2700 molecules of the B protein. Five molecules of the A protein and five molecules of the D protein are located near or at one end of the virion, while ten molecules of the C protein are located near or at the opposite end. The two ends of the phage can be separated by reacting phage fragments, which have been generated by passage of intact phage through a French press, with antibody directed against the A protein (Grant et al., 1981a). By hybridizing the DNA isolated from either end of ³²P-labeled phage to specific restriction fragments of fl replicative form I DNA, we have determined that the single-stranded DNA of the filamentous bacteriophage f1 is oriented within the virion. For wild-type phage, the DNA that codes for the gene III protein is located at the A and D protein end and that which corresponds to the intergenic region is located close to the C protein end of the particle. The intergenic region codes for no protein but contains the origins for both viral and complementary strand DNA synthesis. Analysis of the DNA orientation in phage in which the plasmid pBR322 has been inserted into different positions within the intergenic region of fl shows that the C protein end of all sizes of filamentous phage particles appears to contain a common sequence of phage DNA. This sequence is located near the junction of gene IV and the intergenic region, and probably is important for normal packaging of phage DNA into infectious particles. There appears to be no specific requirement for the origins of viral and complementary strand DNA synthesis to be at the end of a phage particle.     

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.     

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.     

Translational control of bacteriophage f1 gene II and gene X proteins by gene V protein

The gene II region of bacteriophage f1 DNA codes for two proteins, the 46 kd gene II protein and the 13 kd gene X protein, which results from an in-phase start at codon 300 of gene II. Using antigens II protein IgG, we show that the intracellular concentration of both proteins is controlled by the phage gene V protein. In wild-type f1-infected cells, the amount of gene II protein reaches a plateau of about 1500 molecules per cell at 20 min after infection, as measured by blot immunoassay. Similarly, the amount of gene X protein reaches a peak of about 500 molecules per cell around 10 min after infection. In contrast, when the gene V protein is inactive, both gene II and gene X proteins continue to accumulate at a high rate for at least 40 min after infection. This difference is caused by decreased synthesis of gene II and gene X proteins in the presence of gene V protein, which represses the translation of these two proteins.     

The replication of bacteriophage f1: Gene V protein regulates the synthesis of gene II protein

Two filamentous phage gene products are required for the replication of phage DNA. One of these, the gene II protein, is a site-specific endonuclease required for all phage-specific DNA synthesis. The other, the gene V protein, is a single-stranded DNA-binding protein required only for single-strand synthesis. Purified gene V protein, when added to an in vitro protein synthesizing system programmed by f1 DNA, specifically inhibits the synthesis of gene II protein. Inhibition seems to be translational, since synthesis of gene II protein from an RNA template is also inhibited by gene V protein. Gene V protein control of gene II expression can account for the regulation of the level of expression of the filamentous phage genome.     

Reversible denaturation of the gene V protein of bacteriophage f1

The guanidine hydrochloride (GuHCl)-induced denaturation of the gene V protein of bacteriophage f1 has been studied, using the chemical reactivity of a cysteine residue that is buried in the folded protein and the circular dichroism (CD) at 211 and 229 nm as measures of the fraction of polypeptide chains in the folded form. It is found that this dimeric protein unfolds in a single cooperative transition from a folded dimer to two unfolded monomers. A folded, monomeric form of the gene V protein was not detected at equilibrium. The kinetics of unfolding of the gene V protein in 3 M GuHCl and the refolding in 2 M GuHCl are also consistent with a transition between a folded dimer and two unfolded monomers. The GuHCl concentration dependence of the rates of folding and unfolding suggests that the transition state for folding is near the folded conformation.     

G4 DNA replication: I. Origin of synthesis of the viral and complementary DNA strands

The break in the complementary DNA strand of early G4 replicative form II DNA (RFII) and in the viral DNA strand of late RFII DNA was located using two single cleavage restriction enzymes (EcoRI and PstI) and by limited nick translation of the break using DNA polymerase I and ³²P-labelled deoxyribonucleotides followed by digestion with the restriction enzymes HaeIII and HindII. The break in the complementary DNA strand was unique and in HaeIII Z5 close to the EcoRI cleavage site whereas the break in the viral DNA strand was on the other side of the molecule in HaeIII Z2 approxiately 50% away from the EcoRI cleavage site. Distribution of a short ³H pulse in early G4 replicating intermediates that were synthesising both DNA strands at the same time showed that synthesis of the strands started on opposite sides of the molecule and proceeded in opposite convergent directions, suggesting that initiation of synthesis of the two strands was independent and not unified in a single growing fork.     

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.     

Replication of a plasmid containing two origins of bacteriophage f1

A chimeric plasmid was constructed that contains a tandem duplication of the bacteriophage f1 origin of DNA replication. This plasmid replicates stably in the absence of phage. When cells carrying this plasmid are infected with f1, two new plasmid-derived DNA species are generated: a smaller, chimeric plasmid containing only one f1 origin of replication, and a miniphage, the genome of which consists of the f1 fragment that was located between the two f1 origins of the original plasmid. These data support the hypothesis (Horiuchi, 1980) that the nucleotide sequence recognized for initiation of plus strand synthesis in f1 DNA replication is also the signal for its termination.     

A genetic selection for temperature-sensitive variants of the gene V protein of bacteriophage f1.

Complementary negative and positive genetic selections based on the activity of a plasmid-encoded bacteriophage f1 gene V are developed. The negative selection is based on an activity of the gene V protein in E. coli cells which markedly reduces the infection of those cells by f1-related viruses. In order to select against cells expressing active gene V protein, the cells are infected with the p'age R386, a derivative of f1 which confers resistance to chloramphenicol, and are plated in the presence of the antibiotic. Those cells which contain gene V protein are infrequently infected with the virus and are unable to grow in the presence of chloramphenicol; those which do not contain the gene V protein are readily infected and can grow in the presence of the antibiotic. The positive genetic selection consists of excising the gene V sequences from the plasmids and using them to replace the gene V of a bacteriophage f1 derivative containing an amber mutation in gene V. Only those genes which encode an active gene V protein can support phage growth and yield plaques. The two genetic selections can be combined in order to yield a substantial enrichment for genes encoding temperature-sensitive gene V proteins.     

In vitro binding of the bacteriophage f1 gene V protein to the gene II RNA-operator and its DNA analog.

We have investigated the binding of the f1 single-stranded DNA-binding protein (gene V protein) to DNA oligonucleotides and RNA synthesized in vitro. The first 16 nucleotides of the f1 gene II mRNA leader sequence were previously identified as the gene II RNA-operator; the target to which the gene V protein binds to repress gene II translation. Using a gel retardation assay, we find that the preferential binding of gene V protein to an RNA carrying the gene II RNA-operator sequence is affected by mutations which abolish gene II translational repression in vivo. In vitro, gene V protein also binds preferentially to a DNA oligonucleotide whose sequence is the DNA analog of the wild-type gene II RNA-operator. Therefore, the gene V protein recognizes the gene II mRNA operator sequence when present in either an RNA or DNA context.     

Isolation and in vitro characterization of temperature-sensitive mutants of the bacteriophage f1 gene V protein

In vivo selections were used to isolate 43 temperature-sensitive gene V mutants of the bacteriophage f1 from a collection of mutants constructed by saturation mutagenesis of the gene. The sites of temperature-sensitive substitutions are found in both the beta-sheets and the turns of the protein, and some sites are exposed to the solvent while others are not. Thirteen of the variant proteins were purified and characterized to evaluate their free energy changes upon unfolding and their affinities for single-stranded DNA, and eight were tested for their tendencies to aggregate at 42 degrees C. Each of the three temperature-sensitive mutants at buried sites and six of ten at surface sites had free energy changes of unfolding substantially lower (less stabilizing) than the wild-type at 25 degrees C. A seventh mutant at a surface site had a substantially altered unfolding transition and its free energy of unfolding was not estimated. The affinities of the mutant proteins for single-stranded DNA varied considerably, but two mutants at a surface site, Lys69, had much weaker binding to single-stranded DNA than any of the other mutants, while two mutants at another surface site, Glu30, had the highest DNA-binding affinities. The wild-type gene V protein is stable at 42 degrees C, but six of the eight mutants tested aggregated within a few minutes and the remaining two aggregated within 30 minutes at this temperature. Overall, each of the temperature-sensitive proteins tested had a tendency to aggregate at 42 degrees C, and most also had either a low free energy of unfolding (at 25 degrees C), or weak DNA binding. We suggest that any of these properties can lead to a temperature-sensitive gene V phenotype.     

Interaction between gene II protein and the DNA replication origin of bacteriophage f1

The origin of DNA replication of the filamentous bacteriophage f1 binds its initiator protein (gene II protein) in vitro to form a complex that can be trapped on nitrocellulose filters. The binding occurs with both superhelical form DNA and linear DNA fragments. A number of defective mutants of the origin were tested for the ability to bind gene II protein. The region of DNA required for the binding is around a second palindrome downstream from the palindrome that contains the DNA replication initiation site. It overlaps, but is not identical to, the region required for the nicking reaction by the protein. The nicking site itself was dispensable for the binding. In vivo, a number of defective deletion mutants of the origin, when in a plasmid, inhibited growth of superinfecting phage if the intracellular level of gene II protein was low. In addition, these defective origins inhibited the activity of the functional phage origin located on the same replicon. The domain of the DNA sequence required for inhibition in vivo was consistent with that for the binding in vitro.     

Regulation of coliphage f1 single-stranded DNA synthesis by a DNA-binding protein

Control of single-strand DNA synthesis in coliphage f1 was studied with the use of mutants which are temperature sensitive in gene 2, a gene essential for phage DNA replication. Cells were infected at a restrictive temperature with such a mutant, and the DNA synthesized after a shift to permissive temperature was examined. When cells were held at 42 °C for ten or more minutes after infection, only single-stranded DNA was synthesized immediately after the shift to permissive temperature. This indicated that the accumulation of a pool of double-stranded, replicative form DNA molecules is not an absolute requirement for the synthesis of single-stranded DNA, although replicative form DNA accumulation precedes single-strand synthesis in cells infected with wild-type phage. Cells infected at restrictive temperature with the mutant phage do not replicate the infecting DNA, but do accumulate a substantial amount of gene 5 protein, a DNA-binding protein essential for single-strand synthesis. It is proposed that this accumulated gene 5 protein, by binding to the limited number of replicating DNA molecules formed following the shift to the permissive temperature, acts to prevent the synthesis of double-stranded replicative form DNA, thus causing the predominant appearance of single strands. This explanation implies an intermediate common to both single and double-stranded DNA synthesis. The kinetics of gene 5 protein synthesis indicates that it is the ratio of the gene 5 protein to replicating DNA molecules which determines whether an intermediate will synthesize double or single-stranded DNA.     

Studies on the mechanism of enzymatic DNA elongation by Escherichia coli DNA polymerase II.

The mechanism of enzymatic elongation by Escherichia coli DNA polymerase II of a DNA primer, which is annealed to a unique position on the bacteriophage fd viral DNA, has been studied. The enzyme is found to dissociate from the substrate at specific positions on the genome which act as “barriers” to further primer extension. It is believed these are sites of secondary structure in the DNA. When the template is complexed with E. coli DNA binding protein many of these barriers are eliminated and the enzyme remains associated with the same primer-template molecule during extensive intervals of DNA synthesis. Despite the presence of E. coli DNA binding protein, at least one barrier on the fd genome remains rate-limiting to chain extension and disturbs the otherwise processive mechanism of DNA synthesis. This barrier is overcome by increasing the concentration of enzyme.In contrast, it is found that DNA polymerase I is not rate-limited by structural barriers in the template, however, it exhibits a non-processive mechanism of elongation.These findings provide a framework for understanding the necessity for participation of proteins other than a DNA polymerase in chain extension during chromosomal replication.     

Precise localization of the origin of replication in a physical map of HeLa cell mitochondrial DNA and isolation of a small fragment that contains it

The precise positions of the origin of replication³ and of the D-loop within the HpaII restriction map of HeLa cell mitochondrial DNA have been investigated. For this purpose, 7 S DNA, which is the heavy-chain initiation sequence, was used as a template for fragment-primed DNA synthesis by Escherichia coli DNA polymerase I. The results indicate clearly that the origin of replication lies in HpaII fragment 8 at about 80 base-pairs from the border with fragment 17, and that the D-loop region extends from this site, through fragment 17, to a position in fragment 10 which is about 365 base-pairs from the border with fragment 17. Sequential digestion of fragment 8 with HaeIII enzyme has allowed the isolation of a subfragment, about 200 base-pairs long, that contains the origin of replication.     

Bacteriophage f1 DNA replication genes. II. The roles of gene V protein and gene II protein in complementary strand synthesis

Filamentous phage gene V, which encodes a single-stranded DNA binding protein, has been cloned and placed under control of the lac promoter. Cells bearing the clone are refractory to filamentous phage infection if the expression of the gene is induced with isopropyl-1-thio-beta-D-galactoside. The inhibition of infection is shown to occur at an early stage, and can be reversed if the cells express gene II in addition to gene V protein. These observations support the hypothesis that gene II protein, in addition to its role in nicking and facilitating the synthesis of phage viral (+) strand DNA, functions to prevent the gene V-mediated inhibition of complementary (-) strand synthesis. We proposed a model in which the absolute and relative concentrations of the products of genes II, X and V determine whether a single strand is to be exported as phage or incorporated into double-stranded replicative form DNA.