Packaging and Maturation of DNA of Bacteriophage T7 In Vitro

Abstract

We have developed an in vitro complementation assay to demonstrate packaging and maturation of DNA of phageT7. Cells of Escherichia coli B infected with an appropriate T7 amber mutant are concentrated 200-fold and iysed by freezing and thawing. Two extracts from cells infected with different amber mutants are mixed and incubated at 300. Positive complementation results in a 100-fold increase in phage titer.Using this assay we have demonstrated the packaging of phage DNA from an extract that contains no phage heads (gene 9-, 10-), within head structures present in an extract that contains no phage DNA (gene 5-). We have also demonstrated an activity in extracts that contain no phage DNA or heads (gene 5-,9, 10-), which complements gene 19-infected cells. We have proven that this activity is due to the gene-19 product by showing that the activity is temperature-sensitive if the extract is made from cells infected with a mutant having a temperature-sensitive mutation in gene 19. This assay should be useful in elucidating the mechanism of packaging and maturation of DNA of phage T7.In recent years there has been rapid progress in our understanding of the mechanisms by which complex bacteriophages are assembled. Thus, for bacteriophage T4, we know a great deal about the pathways of assembly of tail fibers (1-4), baseplates, sheaths, and cores of the tail (5-9) and assembly of the head (10). These advances have been aided greatly by the development by Edgar and Wood of methods for performing these reactions in cell-free extracts (11,12).The DNA of several bacteriophages (T4, T7, X, and P22) has been shown to replicate as molecules that are several times longer than the DNA found inside the head of the mature virus (13)(14)(15)(16)(17). It is also known that maturation of these long chains of DNA to monomeric units is closely coupled to the formation of the phage head, since cells infected with mutants that are defective in head formation accumulate these long molecules of immature DNA (18-21).However, our knowledge of the molecular mechanisms whereby the immature phage DNA enters the head ("DNA packaging") and is cleaved to monomeric length ("DNA maturation") has been limited, in part, by the inability to perform these reactions in vitro. Recently, however, assays have been developed to demonstrate packaging of phage DNA in vitro. Kaiser and Masuda (22) showed that exogenous lambda DNA could be packaged by extracts from induced lysogens and Hohn and Hohn (23) have demonstrated that head-related particles containing no DNA ("petit lambda") can be filled with DNA in vitro. Finally, the experiments of Pruss, Goldstein, and Calendar (24) showed that the size of the head of phage P2 is determined solely by the proteins involved and not by the phage DNA.Bacteriophage T7 may be a convenient system in which to study DNA packaging and maturation because it is a 3545 relatively simple virus for which there is a great deal of information about its genetics and physiology (25). The DNA of phage T7 is a unique, terminall...

In vitro repair of double-strand breaks accompanied by recombination in bacteriophage T7 DNA

“…Other workers have reported in vitro recombination of T7 DNA (6,9,(25)(26)(27)(28)(29)42). Recombination has also been observed with an in vitro system designed to maximize DNA replication (14,15).…”

“…The precise molecular mechanisms responsible for T7 recombination are poorly understood. Therefore, possible involvement of strand breaks in this phage's recombination mechanism(s) remains an open question.Other workers have reported in vitro recombination of T7 DNA (6,9,(25)(26)(27)(28)(29)42 …”

In vitro DNA Packaging of PRD1: A Common Mechanism for Internal-membrane Viruses

“…In the phi6 system, single-stranded RNA is translocated into a preformed capsid where it is replicated to double-stranded form. [2][3][4] In vitro packaging systems have been developed for tailed dsDNA bacteriophages using slightly different encapsidation mechanisms: (a) phages using terminases cutting and packaging the DNA in a headful manner, such as P22, 5,6 SPP1, 7 T4, 8,9 and T1; 10 (b) phages cutting their concatemeric DNA only at specific cut sites, such as lambda, 11 T3, 12 and T7; 13 and (c) phages having unit-length DNA genomes that are not cleaved during packaging, such as phi29. 14,15 Of these, the packaging of bacteriophage phi29 is probably the best characterised.…”

Purified Membrane-Containing Procapsids of Bacteriophage PRD1 Package the Viral Genome

“…Bacteriophages use terminase to cut the concatemeric DNA when the head is fully packaged (P22, 13,14 SPP1, 15 T4, 16 and T1 17 ), use terminase to cut their concatemeric DNA at specific sites (λ, 18 T3, 19 and T7 20 ), package unit-length genomes with covalently linked terminal proteins (ϕ29 21,22 and PRD1 23 ), sequentially package segmented singlestranded genome precursors (ϕ6 24 ), or package the genome during replication (ϕX174 25 ). To obtain insights into the viral genome packaging encapsidation mechanisms, in vitro packaging systems have been developed for many bacteriophages.…”