Folded, concatenated genomes as replication intermediates of bacteriophage T7 DNA

Abstract

A complex form of bacteriophage T7 DNA, containing up to several hundred phage equivalents of DNA, arises during replication of T7. The complex was stable to treatment with ionic detergent, Pronase, and phenol. The complex form normally exists for only a short time, corresponding to the phase of rapid T7 DNA synthesis. It is then converted to shorter molecules, both concatemers and unit-size DNA. The complex was stable up to the temperature of denaturation of the bihelix. It consisted of a series of loops emanating from a dense central core, as shown by electron microscopy. The complex form is similar to the relaxed Escherichia coli folded chromosome ("nucleoid"). The loops contained an average of 0.7 to 0.8 phage equivalent of DNA. During infection by phage with an amber mutation in gene 3 (endonuclease), formation of the complex occurred normally, but its maturation to unit-size DNA was blocked. Before treatment with phenol, the complex contained short fragments of newly replicated DNA. These were released as single-stranded pieces during phenol treatment. A pathway for T7 DNA replication is indicated in which the flow of material is from unit-size DNA to linear concatemers to the complex form, and then back to unit-size DNA by way of linear concatemers.

Halophage HF2: genome organization and replication strategy

“…The maximum concatamer size appeared to represent six genomic units, although this band was approaching the upper limit of gel resolution and may have included larger genomic concatamers. A considerable amount of material hybridizing with the probe was present in on July 9, 2020 by guest http://jvi.asm.org/ the gel wells, indicating virus-specific DNA that was unable to migrate out of the agarose blocks, possibly because of the formation of complex genomic replication intermediates with poor mobility (8) or branched concatameric complexes (26). A final sample, taken after culture lysis, showed only a monomeric phage-specific band migrating at approximately 80 kb, representing the mature phage genomes (Fig.…”

Purification and structures of recombining and replicating bacteriophage T7 DNA

“…The method for labeling DNA was described earlier (13). E. coli DNA was bulk labeled by adding a total of 1.5 utCi of [14C]thymidine (dThd) per ml (50 mCi/mmol) in several aliquots.…”

“…Cells were collected by centrifugation and suspended in 100 mM Tris (pH 7.5)-50 mM NaCl-10 mM EDTA in a volume Yio that of the original culture. They were incubated for 20 min at 0°C with 0.1 mg of lysozyme per ml and then treated with Sarkosyl, Pronase, and phenol as described earlier (13).…”

“…Characterization methods. Neutral sucrose gradients were made up and run as described previously (13). All gradients were run in a Beckman SW50.1 rotor at 4°C.…”

“…This approach requires a preparative density gradient and suffers from the disadvantage that some DNA structures may be lost or altered in the CsCl gradient. For example, the large flowerlike structures that we recently characterized as intermediates of T7 replication (13) were not observed in the DNA isolated from CsCl gradients after density shift experiments (R. C.…”