Genetic deletions between directly repeated sequences in bacteriophage T7

Summary DNA sequence analysis of genetic deletions in bacteriophage T7 has shown that these chromosomal rearrangements frequently occur between directly repeated DNA sequences. To study this type of spontaneous deletion in more quantitative detail synthetic fragments of DNA, made by hybridizing two complementary oligonucleotides, were introduced into the non-essential T7 gene 1.3 which codes for T7 DNA ligase. This insert blocked synthesis of functional ligase and made the phage that carried an insert unable to form plaques on a host strain deficient in bacterial ligase. The sequence of the insert was designed so that after it is put into the T7 genome the insert is bracketed by direct repeats. Perfect deletion of the insert between the directly repeated sequences results in a wild-type phage. It was found that these deletion events are highly sensitive to the length of the direct repeats at their ends. In the case of 5 bp direct repeats excision from the genome occurred at a frequency of less than 10⁻¹⁰, while this value for an almost identical insert bracketed by 10 bp direct repeats was approximately 10⁻⁶. The deletion events were independent of a hostrecA mutation.     

Deletion between direct repeats in T7 DNA stimulated by double-strand breaks.

An in vitro system based on extracts of Escherichia coli infected with bacteriophage T7 was used to study genetic deletions between directly repeated sequences. The frequency of deletion was highest under conditions in which the DNA was actively replicating. Deletion frequency increased markedly with the length of the direct repeat both in vitro and in vivo. When a T7 gene was interrupted by 93 bp of nonsense sequence flanked by 20-bp direct repeats, the region between the repeats was deleted in about 1 out of every 1,600 genomes during each round of replication. Very similar values were found for deletion frequency in vivo and in vitro. The deletion frequency was essentially unaffected by a recA mutation in the host. When a double-strand break was placed between the repeats, repair of this strand break was often accompanied by the deletion of the DNA between the direct repeats, suggesting that break rejoining could contribute to deletion during in vitro DNA replication.     

Effects of Na+ on the persistence length and excluded volume of T7 bacteriophage DNA.

Total intensity, Rayleigh light scattering has been used to measure the rms radius, second virial coefficient, persistence length, and excluded volume of homogeneous T7 bacteriophage DNA as a function of Na+ concentration (0.005 to 3.0 M). All parameters decrease sharply as [Na+] increases, and tend to level off at high Na+. The variation of persistence length with [Na+] is consistent with predictions from counterion condensation theory.     

High-frequency spontaneous deletion of DNA sequences flanked by direct DNA repeats which also contain an internal palindrome

The DNA sequences associated with a very high-frequency, spontaneous deletion event have been determined to be two 11-base direct repeats which also contain an internal 6-base palindrome. A parental M13 replicative form (RF) DNA harboring DNA fragments of the T4 denV gene contained these direct repeats and could only be maintained at 5% of the total RF DNA within an infected cell. The remaining RF DNA was deleted for all intervening sequences between the direct repeats (2.2-kb), but one copy of the direct repeat was retained after the deletion had occurred. This site-specific deletion was highly reproducible in that if parental-sized M13 RF DNA was gel purified and transformed back into cells, the deletion occurred at precisely the same sequence as before. Electron microscopic analyses of DNA extracted from cells transformed with parental-sized DNA revealed the presence of excised 2.2-kb double-stranded circular DNA molecules. This observation thus rules out a copy choice replication/deletion mechanism to account for this high-frequency deletion event.     

The termini of bacteriophage T7 DNA

The termini of Escherichia coli phage T7 DNA have been labeled with ³²P by the polynucleotide kinase reaction. The DNA was fragmented, denatured, and annealed to denatured T7 DNA embedded in agar; elution was measured as a function of temperature. The terminal fragments were eluted from the gel at temperatures well below that of the bulk of the DNA, suggesting that these regions have a very high adenine-plus-thymine content. However, when DNA doubly labeled throughout at random by growth of the phage in [³H]thymidine and ³²PO₄, was denatured, annealed to the gel, and eluted as a function of temperature, the material eluting from the gel in this low-temperature range was about 50% adenine and thymine. Hence the melting behavior of the terminal fragments is not a result of a high adenine plus thymine content. By electrophoretic analysis of exonucleolytic digests of the T7 DNA it was shown that no unusual bases were present. It is suggested that the low thermal stability of the annealed terminal fragments is a consequence of the high guanine·cytosine regions being unavailable for hybridization, possibly because they are involved in intra-strand hydrogen bonding.     

Binding of ethidium to bacteriophage T7 and T7 deletion mutants

Equilibrium binding of ethidium, quantitated by fluorescence enhancement, to DNA packaged in bacteriophage T7 and T7 deletion mutants has been compared with the binding of this dye to DNA released from its capsid (free DNA). During achievement of apparent equilibrium binding, no change in bacteriophage T7 structure occurred, by the criterion of agarose gel electrophoresis. However, excessive incubation with ethidium bromide caused detectable changes in bacteriophage structure, a possible explanation of disagreements in similar studies previously performed with T-even bacteriophages. Scatchard plots for packaged DNA had a curvature greater than the previously demonstrated [Bresloff, J. L. & Crothers, D. M. (1981) Biochemistry 20 , 3547–3553] curvature for free DNA. By treating plots for packaged DNA as though they were biphasic, it was found that binding to most sites occurred with an apparent association constant (K_ap) 3.3–4.3 times lower than the K_ap of free DNA. The number of these sites increased significantly as the density of packaged DNA was decreased by use of the deletion mutants. Values of ΔH° for these sites were negative and equal to the ΔH° for free DNA; values of ΔS° were positive and about half the ΔS° for free DNA. A second class of sites, roughly 1.2% of the total, had a significantly higher K_ap and more negative ΔH° than those of the majority of sites.     

DNA injection and genetic recombination of alkylated bacteriophage T7 in the presence of nalidixic acid.

Marker rescue experiments with alkylated T7 bacteriophage carried out in the presence and in the absence of nalidixic acid suggest that the gradient in rescue is due to two alkylation-induced causes: a DNA injection defect and an interference with DNA synthesis.     

A molecular handoff between bacteriophage T7 DNA primase and T7 DNA polymerase initiates DNA synthesis

The T7 DNA primase synthesizes tetraribonucleotides that prime DNA synthesis by T7 DNA polymerase but only on the condition that the primase stabilizes the primed DNA template in the polymerase active site. We used NMR experiments and alanine scanning mutagenesis to identify residues in the zinc binding domain of T7 primase that engage the primed DNA template to initiate DNA synthesis by T7 DNA polymerase. These residues cover one face of the zinc binding domain and include a number of aromatic amino acids that are conserved in bacteriophage primases. The phage T7 single-stranded DNA-binding protein gp2.5 specifically interfered with the utilization of tetraribonucleotide primers by interacting with T7 DNA polymerase and preventing a productive interaction with the primed template. We propose that the opposing effects of gp2.5 and T7 primase on the initiation of DNA synthesis reflect a sequence of mutually exclusive interactions that occur during the recycling of the polymerase on the lagging strand of the replication fork.     

The context analysis of polynucleotide sequences. II. Inverted repeats and complementary palindromes in RNA-polymerase genes

A new approach to the reconstruction of the RNA secondary structure is suggested on the basis of the method of contextual analysis of polynucleotide sequences. The coding gene regions of beta-, beta'-, sigma-subunits of E. coli RNA polymerase and of phage T7 RNA polymerase were analysed. The clusters of non-random inverted repeats were found in all these genes. The mRNA coded by them can be folded into compact secondary structures. The latter are formed by quite long helices with a few cases of mispairing.     

Mutagenesis of bacteriophage T7 and T7 DNA by alkylation damage.

We have developed a new assay for in vitro mutagenesis of bacteriophage T7 DNA that measures the generation of mutations in the specific T7 gene that codes for the phage ligase. This assay was used to examine mutagenesis caused by in vitro DNA synthesis in the presence of O6-methylguanosine triphosphate. Reversion of one of the newly generated ligase mutants by ethyl methanesulfonate was also tested.     

Sedimentation and viscosity of bacteriophage T7 DNA in presence of CH3HgOH

The effects of increasing concentartions of methylmercuric hydroxide (CH₃HgOH) on the rate of sedimentation, S⁰, and intrinsic viscosity, [η], of T7 DNA have been studied at 20°C in 0.005, 0.05, and 0.5M Na₂SO₄, respectively, whereby each salt solvent conatined, in addition, 0.005M sodium borate, pH 9.18, as a buffer. Both S⁰ and [η] are independent of organomercurial concentration as long as DNA remains native. Denaturation, brought about by the complexing of CH₃HgOH with the polymer, produces large changes in S⁰ as wll as [η]. The sedimentation coefficient increases strongly with increasing oragnomercurial concentration once strand separation has occured. Experimental difficulties prevented measuring of [η] in the posttransition region. The data on S⁰ have been used, in combination with available information on the so-called density increment (?ρ/?c₂), to obtain information on the frictional properties of single-stranded and methylmercurated T7 DNA. The frictional coefficient, defined as f′₂ = M₂(?p/?c₂)/S⁰ηN_A, where M₂ is the molecular wieght of T7 DNA, c₂ is the concentration of DNA in g/ml of solution, η_r the realtive viscosity of the salt solvents, and N_A is Avogadro's number, was evaluated for all three salt media as a function of organomercurial concentration. f′₂ of native T7 DNA was found not to be sensitive to changes in ionic strngth; but f′₂ of single-stranded and methylmercurated T7 DNA varied strongly with salt concentration. Since f′₂ of single-stranded T7 DNA was barely affected by organomercurial concentration at a given ionic strength, it is concluded that the dramatic variations of S⁰ with pM (pM ≡ -log[CH₃HgOH]) observed in the posttransition zone reflect only changes in the thermodynamic interactions (“preferential interactions”) existing between DNA and the vatious other solution components, but not changes in the coil dimensions of the polymer.     

Distribution of pyrimidine sequences of different length and base composition in bacteriophage T5 DNA]

Distribution of pyrimidine tracts of different length (isopliths) with general formula PynPn+1 in bacteriophage T5 DNA was studied. The first seven isoplith fractions were subfractionated by the chain length and the quantity of the resulting non-isomeric oligonucleotides was determined. The pattern of distribution of pyrimidine tracts of various length and base composition in bacteriophage T5 DNA is different from that previously observed in the DNAs of bacteriophages T3 and T7. The observed differences in distribution of pyrimidine nucleotides are in accordance with the other peculiarities of bacteriophage T5 genome.     

Involvement of DNA gyrase in bacteriophage T7 growth.

We have found that the burst size of bacteriophage T7 was decreased in two Escherichia coli temperature-sensitive gyrase mutants incubated at the restrictive temperature. This reduction in burst size indicates that gyrase may be required for T7 growth.     

The terminal redundant regions of bacteriophage T7 DNA

Summary Some aspects of the involvment of the terminal reduntant regions of T7 DNA on phage production have been studied by transfection experiments with T7 DNA after treatment of the molecules with exonuclease or exonuclease plus exonuclease I. It was found that terminal 5 gaps between 0.08 and 6.4% of the total length did not decrease the infectivity of the molecules although such gaps cannot be filled directly by DNA polymerases. Rather, compared to fully native DNA the infectivity of gapped DNA increased up to 20 fold in rec ⁺ spheroplasts and up to 4 fold in recB spheroplasts. This indicates a protective function of the single-stranded termini against the recBC enzyme in rec ⁺ and possibly another unidentified exonuclease present also in recB. The possibility that spontaneous circularization of the gapped molecules in vivo provides protection against exonucleolytic degradation was tested by transfection with T7 DNA circularization in vitro by thermal annealing. Such molecules were separated from linear molecules by neutral sucrose gradient centrifugation. They displayed a 3 to 6 fold higher infectivity in rec ⁺ and recB compared to linear gapped molecules, which shows that T7 phage production may effectively start from circular DNA.When the 3 single-stranded ends from gapped molecules were degraded by treatment with exonuclease I the infectivity of the molecules was largely abolished in rec ⁺ and recB as soon as 40 to 80 base pairs had been removed per end. It is concluded that the terminal regions of T7 DNA molecules are essential for phage production and that the redundancy comprises probably considerably less than 260 base pairs. The results are discussed with respect to the mode of T7 DNA replication.     

Specific deletion of DNA sequences between preselected bases.

Blunt-end ligation of a "filled-in" HindIII, Sal I, Ava I or Bcl I restriction site with a DNA fragment having A, G, C, or T as the terminal 3' nucleotide regenerates the corresponding restriction site. A combination of this property with the action of BAL 31 nuclease which progressively removes base-pairs from the ends of linear DNA, can generate deletions extending to desired pre-selected nucleotides, and introduces unique restriction sites at those positions. Similarly other restriction sites can be used to select for the deletion of sequences between specific di-, tri-, tetra- and penta-nucleotides. Using this method, 10 base pairs were deleted from the end of a restriction fragment carrying the late promoter for bacteriophage T7 gene 1.1, to create a molecule with a unique restriction site at the initiation codon for translation.     

Intracellular organization of bacteriophage T7 DNA: analysis of parenteral bacteriophage T7 DNA-membrane and DNA-protein complexes.

After infection of Escherichia coli with bacteriophage T7, the parenteral DNA forms a stable association with host cell membranes. The DNA-membrane complex isolated in cesium chloride gradients is free of host DNA and the bulk of T7 RNA. The complex purified through two cesium chloride gradients contains a reproducible set of proteins which are enriched in polypeptides having molecular weights of 54,000, 34,000, and 32,000. All proteins present in the complex are derived from host membranes. Treatment of the complex with Bruij-58 removes 95% of the membrane lipid and selectively releases certain protein components. The Brij-treated complex has an S value of about 1,000 and the sedimentation rate of this material is not altered by treatment with Pronase or RNase.