Bacteriophage T7 DNA Packaging Research Articles

Nano-channel of viral DNA packaging motor as single pore to differentiate peptides with single amino acid difference

Detection, differentiation, mapping, and sequencing of proteins are important in proteomics for the assessment of cell development such as protein methylation or phosphorylation as well as the diagnosis of diseases including metabolic disorder, mental illness, immunological ailments, and malignant cancers. Nanopore technology has demonstrated the potential for the sequencing or sensing of DNA, RNA, chemicals, or other macromolecules. Due to the diversity of protein in shape, structure and charge and the composition versatility of 20 amino acids, the sequencing of proteins remains challenging. Herein, we report the application of the channel of bacteriophage T7 DNA packaging motor for the differentiation of an assortment of peptides of a single amino acid difference. Explicit fingerprints or signatures were obtained based on current blockage and dwell time of individual peptide. Data from the clear mapping of small proteins after protease digestion suggests the potential of using T7 motor channel for proteomics including protein sequencing.

Biophysical Studies Reveal New Evidence for One-Way Revolution Mechanism of Bacteriophage ϕ29 DNA Packaging Motor

Biophysical Studies Reveal New Evidence for One-Way Revolution Mechanism of Bacteriophage ϕ29 DNA Packaging Motor

Formation and cleavage of a DNA network during in vitro bacteriophage T7 DNA packaging: light microscopy of DNA metabolism.

To understand in vivo DNA metabolism, in vitro systems are developed that perform DNA metabolism, while maintaining in vivo (physiological) character. To determine the state of DNA during in vitro physiological metabolism, the present study develops procedures of fluorescence light microscopy for observation of stained DNA molecules during in vitro physiological metabolism in a crude extract of bacteriophage T7-infected cells. The extract inhibits illumination-induced breakage of DNA. The following DNA metabolism remains active for 2-3 min during microscopy: exonuclease-dependent end-to-end joining (concatemerization) of T7 DNA and subsequent cleavage of concatemers. When the T7 gene 3-encoded DNA debranching endonuclease is absent during in vitro T7 DNA concatemerization, DNA progressively partitions to form a continuous, mostly immobile (i.e., no detected Brownian motion) fibrous network that encloses the DNA-depleted solution; presumably because of reduced branching, a less extensive network forms when the gene 3-encoded debranching endonuclease is present. Most strands of the network consist of multiple DNA segments. After a time interval of 5-10 min, the DNA network undergoes cleavage that depends on the presence of both ATP, capsids, and the DNA packaging accessory proteins encoded by genes 18 and 19; multiple cleavages eventually disrupt the continuity of the DNA network. The dependence of the observed cleavage on these factors is explained by the hypothesis that this cleavage is the first of two cleavages known to occur during the packaging of T7 DNA concatemers both in vivo and in vitro. The first cleavage is also known to initiate entry of DNA into a T7 capsid. The cleavage observed here is usually preceded by an approximately 10 s burst of oscillatory motion of the DNA network near the point of eventual cleavage. If the in vivo presence of a similar concatemer-containing DNA network is assumed, requirement for DNA packaging-associated release of DNA from this network is a possible explanation for the evolution of a T7 DNA packaging pathway that is initiated by cleavage of a concatemer.

The Direction and Rate of Bacteriophage T7 DNA Packaging in Vitro

To determine the direction of the entry of DNA during in vitro bacteriophage T7 DNA packaging, incompletely packaged DNA (ipDNA) was fractionated by agarose gel electrophoresis after degradation of DNA outside of capsids and then release of packaged DNA from capsids. After fractionation, quantitative in-gel probing with a right end-specific oligonucleotide detects heterogeneous ipDNA (called right-end ipDNA). Most of the right-end ipDNA appears with kinetics expected of a precursor to the mature T7 DNA. In-gel probing with a left-end-specific oligonucleotide detects ipDNA (left-end ipDNA); the molar amount of left end ipDNA is always at least 50× less than the molar amount of right-end ipDNA. Left-end ipDNA appears with the kinetics of an abortive end product of T7 DNA packaging. Thus, productive T7 DNA packaging occurs in a right-to-left direction. Quantitation of the conversion of right-end ipDNA to mature-length DNA yields an estimate of the mean rate of right-to-left in vitro T7 DNA packaging: 28 ± 6 kbp/min for the last 20-50% of the DNA packaged.

Role of exonuclease in the specificity of bacteriophage T7 DNA packaging

During morphogenesis in vivo, bacteriophage T7 packages and cuts to mature size an end-to-end concatemer of its nonpermuted, terminally repetitious, double-stranded, mature DNA. Efficient production (90–100%) and packaging (20–35%) of concatemers has also been demonstrated in extracts of T7-infected cells ( in vitro) (Son, M., Hayes, S. J., and Serwer, P. [1988] Virology 162, 38–46). By use of both this procedure of in vitro DNA packaging and in-gel hybridization to packaged DNA fractionated by agarose gel electrophoresis, the specificity of packaging in vitro is found to depend on the presence of T7 gene 6 exonuclease (p6). In the absence of p6 in vitro, no concatemerization is detected and packaging of DNA nonhomologous to T7 DNA (bacteriophage P22 DNA) is as efficient (0.05–1.10/6) as the packaging of monomeric T7 DNA. Addition of p6 in vitro both stimulates the concatemerization-packaging of T7 DNA and suppresses the packaging of P22 DNA. The packaging efficiency for concatemeric T7 DNA is 29–611 × higher than that for monomeric T7 DNA. Inhibition of the packaging of P22 DNA by p6 is correlated with the formation of single-stranded P22 DNA ends. These data are explained by the hypothesis that a DNA molecule with a single-stranded end is packaged less efficiently than the same DNA without the single-stranded end. Testing this hypothesis in vivo reveals that both p6 and gene 3 endonuclease contribute to suppressing the packaging of host DNA.

Bacteriophage T7 DNA Packaging: I. Plasmids containing a T7 replication origin and the T7 concatemer junction are packaged into transducing particles during phage infection

Bacteriophage T7 DNA is a linear duplex molecule with a 160 base-pair direct repeat (terminal redundancy) at its ends. During replication, large DNA concatemers are formed, which are multimers of the T7 genome linked head to tail through recombination at the terminal redundancy. We define the sequence that results from this recombination, a mature right end joined to the left end of T7 DNA, as the concatemer junction. To study the processing and packaging of T7 concatemers into phage particles, we have cloned the T7 concatemer junction into a plasmid vector. This plasmid is efficiently (at least 15 particles/infected cell) packaged into transducing particles during a T7 infection. These transducing particles can be separated from T7 phage by sedimentation to equilibrium in CsCl. The packaged plasmid DNA is a linear concatemer of about 40 x 10(3) base-pairs with ends at the expected T7 DNA sequences. Thus, the T7 concatemer junction sequence on the plasmid is recognized for processing and packaging by the phage system. We have identified a T7 DNA replication origin near the right end of the T7 genome that is necessary for efficient plasmid packaging. The origin, which is associated with a T7 RNA polymerase promoter, causes amplification of the plasmid DNA during T7 infection. The amplified plasmid DNA sediments very rapidly and contains large concatemers, which are expected to be good substrates for the packaging reaction. When cloned in pBR322, a sequence containing only the mature right end of T7 DNA is sufficient for efficient packaging. Since this sequence does not contain DNA to the right of the site where a mature T7 right end is formed, it was expected that right ends would not form on this DNA. In fact, with this plasmid the right end does not form at the normal T7 sequence but is instead formed within the vector. Apparently, the T7 packaging system can also recognize a site in pBR322 DNA to produce an end for packaging. This site is not recognized solely by a "headful" mechanism, since there can be considerable variation in the amount of DNA packaged (32 x 10(3) to 42 x 10(3) base-pairs). Furthermore, deletion of this region from the vector DNA prevents packaging of the plasmid. The end that is formed in vector DNA is somewhat heterogeneous. About one-third of the ends are at a unique site (nucleotide 1712 of pBR322), which is followed by the sequence 5'-ATCTGT-3'. This sequence is also found adjacent to the cut made in a T7 DNA concatemer to produce a normal T7 right end.

Bacteriophage T7 DNA packaging: III. A “Hairpin” end formed on T7 concatemers may be an intermediate in the processing reaction

An unusual left end (M-end) has been identified on bacteriophage T7 DNA isolated from T7-infected cells. This end has a "hairpin" structure and is formed at a short inverted repeat sequence centered around nucleotide 39,587 of T7, 190 base-pairs to the left of the site where a mature left end is formed on the T7 concatemer. We do not detect the companion right end that would be formed if the M-end is produced by a double-stranded cut on the T7 concatemer. This suggests that the hairpin left end may be generated from a single-stranded cut in the DNA that is used to prime rightward DNA synthesis. The formation of M-end does not require the products of T7 genes 10, 18 or 19, proteins that are essential for the formation of mature T7 ends. During infection with a T7 gene 3 (endonuclease) mutant, phage DNA synthesis is reduced and the concatemers are not processed into unit length DNA molecules, but both M-end and the mature right end are formed on the concatemer DNA. These two ends are also found associated with the large, rapidly sedimenting concatemers formed during a normal T7 infection while the mature left end is present only on unit length T7 DNA molecules. We propose that DNA replication primed from the hairpin end produced by a nick in the inverted repeat sequence provides a mechanism to duplicate the terminal repeat before DNA packaging. Packaging is initiated with the formation of a mature right end on the branched concatemer and, as the phage head is filled, the T7 gene 3 endonuclease may be required to trim the replication forks from the DNA. Concatemer processing is completed by the removal of the 190 base-pair hairpin end to produce the mature left end.

Bacteriophage T7 DNA Packaging: II. Analysis of the DNA sequences required for packaging using a plasmid transduction assay

Recombinant plasmids carrying a bacteriophage T7 origin of DNA replication and sequences from the T7 concatemer junction are efficiently packaged into transducing particles during phage infection. With some constructs, as many as 50 transducing particles are produced per infected cell. We have used this plasmid packaging system to determine which T7 DNA sequences are required for the processing and packaging of the plasmid concatemers and to investigate the effects of altering the spacing and orientation of the required sequences. An origin of T7 DNA replication is essential for high-efficiency transduction, presumably to form the plasmid concatemers that are the substrates of the packaging reaction. In addition, two short sequences from the concatemer junction are required, one flanking the site where the right end of T7 DNA is formed (pacR) and the other flanking the site for formation of the left end (pacL). The spacing between pacR and pacL is not important, but the sequences must be positioned in the same orientation on the plasmid. With certain deletions of pacL, the specificity of end formation is reduced but the efficiency of packaging is near normal. Plasmids that contain only one of the two pac sites are packaged at about 10% of the efficiency of those with both sites. The residual packaging of these plasmids results from regeneration of the other packaging site by recombination with T7 phage DNA. To function in plasmid packaging, the sequences from the concatemer junction must be positioned on the plasmid in the same orientation relative to the T7 replication origin as is found in T7 DNA. This apparently results from a requirement for transcription through these sequences in the rightward direction from the T7 promoter that is associated with the replication origin. Such transcription from another T7 promoter (phi 10), that is not itself a replication origin, allows packaging when the origin is in the opposite orientation.

Role of gene 6 exonuclease in the replication and packaging of bacteriophage T7 DNA

When bacteriophage T7 gene 6 exonuclease is genetically removed from T7-infected cells, degradation of intracellular T7 DNA is observed. By use of rate zonal centrifugation, followed by either pulsed-field agarose gel electrophoresis or restriction endonuclease analysis, in the present study, the following observations were made. (1) Most degradation of intracellular DNA requires the presence of T7 gene 3 endonuclease and is independent of DNA packaging; rapidly sedimenting, branched DNA accumulates when both the gene 3 and gene 6 products are absent. (2) A comparatively small amount of degradation requires packaging and occurs at both the joint between genomes in a concatemer and near the left end of intracellular DNA; DNA packaging is only partially blocked and end-to-end joining of genomes is not blocked in the absence of gene 6 exonuclease. (3) Fragments produced in the absence of gene 6 exonuclease are linear and do not further degrade; precursors of the fragments are non-linear. (4) Some, but not most, of the cleavages that produce these fragments occur selectively near two known origins of DNA replication. On the basis of these observations, the conclusion is drawn that most degradation that occurs in the absence of T7 gene 6 exonuclease is caused by cleavage at branches. The following hypothesis is presented: most, possibly all, of the extra branching induced by removal of gene 6 exonuclease is caused by strand displacement DNA synthesis at the site of RNA primers of DNA synthesis; the RNA primers, produced by multiple initiations of DNA replication, are removed by the RNase H activity of gene 6 exonuclease during a wild-type T7 infection. Observation of joining of genomes in the absence of gene 6 exonuclease and additional observations indicate that single-stranded terminal repeats required for concatamerization are produced by DNA replication. The observed selective shortening of the left end indicates that gene 6 exonuclease is required for formation of most, possibly all, mature left ends.

Optimization of the in vitro packaging efficiency of bacteriophage T7 DNA: effects of neutral polymers

The in vitro DNA packaging of several DNA bacteriophages is stimulated by the presence of neutral polymers. To optimize bacteriophage T7 DNA packaging and to understand the basis for optimization, the efficiency ofT7 DNA packaging has been determined at completion, as a function of the type, molecular mass, and concentration of the polymer added. When the polymer used was polyethylene glycol (PEG) of 0.2, 0.6 or 12.6 kDa, the efficiency of DNA packaging reached maximum at an intermediate concentration of polymer. The osmotic pressure ( P os) at maximum efficiency was either in, or close to, the range of colloid P os measured for the intact host cell. The optimum P os increased as the size of the polymer used decreased. PEG-100 (of 0.1 kDa) did not stimulate in vitro T7 DNA packaging. Dextran of 10 kDa also stimulated packaging and produced maximum efficiency at a physiological P os. The degree of stimulation increases as DNA packaging extract concentration decreases; stimulation by as much as two to three orders of magnitude is observed. The presence of added polymer reduces fluctuations in DNA packaging efficiency caused by variability in the concentration of DNA packaging extracts. For reproducible and high efficiency packaging, the dextran was more reliable than the PEGs, possibly because the P os of the dextran solutions is less sensitive to polymer concentration than is the P os of PEG solutions. The optimum concentration of dextran at completion was also the optimum at all times before completion.

Concatemerization and packaging of bacteriophage T7 DNA in vitro: determination of the concatemers' length and appearance kinetics by use of rotating gel electrophoresis

During its morphogenesis both in intact infected cells ( in vivo) and in lysates of infected cells ( in vitro), bacteriophage T7 forms end-to-end concatemers of its mature DNA, a linear, nonpermuted, terminally repetitious DNA. During morphogenesis, in vivo T7 concatemers are packaged in preformed capsids and cut to mature size. In the present study the lengths and appearance kinetics of concatemers formed in vitro from mature T7 DNA have been determined. The following procedures are used here for the first time: (a) 20–35% efficient in vitro concatemerization and packaging of T7 DNA; the mixture used for packaging contained two lysates that together had all T7 gene products, and (b) fractionation of concatemers by rotating gel electrophorsis (RGE), which improves the resolution by length of concatemer-length DNA. Concatemerization at 30° was so fast that some other process must be rate limiting for packaging. The concatemers formed were linear and joined left-end to right-end by complementary base pairing, not by blunt-end ligation. Concatemers formed at 30° were reconverted to mature DNA by packaging in vitro. Reducing the temperature to 0° both slowed concatemerization to the time scale (minutes) needed for control of the extent of concatemerization and reduced packaging to insignificant levels, thereby also uncoupling packaging from concatemerization. At both 30° and 0° bands of discrete-length concatemers were observed by RGE. The lengths were n times the length of mature T7 DNA; n was found to be any integer from 2 to 15. The bands were stronger at 0° than they were at 30° in comparison to a background of heterogeneous DNA. No evidence for the favoring of any value of n was found. In addition, it was found by two-dimensional agarose gel electrophoresis that a comparatively small amount of circular DNA was produced in vitro.

In vitro packaging of bacteriophage T7 DNA requires ATP

Removal of nucleoside triphosphates from extracts prepared from bacteriophage T7-infected Escherichia coli results in a stringent requirement for added ATP to form infective phage particles by in vitro packaging of bacteriophage T7 DNA. Optimal packaging efficiency was achieved at a concentration of about 1.25 mM. Other nucleoside triphosphates could be substituted for ATP, but none of the common nucleoside triphosphates was as effective as ATP in promoting in vitro encapsulation.