Purification and structures of recombining and replicating bacteriophage T7 DNA

Abstract

During the infection of Escherichia coli by bacteriophage T7, there is a gradual conversion of host DNA to T7 DNA. Recombination and replication occur during this time. We have devised a new way of examining the physical structures of the intermediates of these processes. It is based on the observation that there are no sites in T7 DNA susceptible to cleavage by the restriction endonuclease EcoRI. E. coli DNA, on the other hand, is susceptible to degradation by EcoRI. Thus, phage and host DNA can be separated by sucrose gradient centrifugation after treatment with EcoRI. Concatemeric T7 DNA contains a high proportion of branched, gapped, and whiskered structures. These appear to be intermediates of replication and recombination. This approach also monitors the conversion process from host to T7 DNA.

Multidimensional analysis of intracellular bacteriophage T7 DNA: effects of amber mutations in genes 3 and 19

“…Intracellular T7 DNA was then labeled with 3H by adding [3Hlthymidine to the infected cells at the time indicated. T7 stops the synthesis of DNA by its host, and the DNA labeled by this procedure is primarily T7 DNA (21,23). To quench the process of infection, either part or all of an infected culture was mixed with an equal volume of an ice-cold solution that contained 0.3 M NaCl, 0.1 M Tris chloride, pH 7.4, 0.01 M EDTA, 0.008 M KCN, and 50% sucrose.…”

“…In the past, analysis of the DNA in lysates of bacteriophage T7-infected cells has been performed by rate-zonal centrifugation in a density gradient, usually followed by fractionation of the gradient and assay for DNA (21,23,29,37,38,50) (Fig. 1).…”

“…Linear DNAs longer than mature T7 DNA (concatemers) are at least some of the 32S to 100S DNA (16,21,23,37,50). In addition to the 32S to lOOS DNA, a replicating (and therefore branched) DNA that appears collapsed when viewed by electron microscopy and that sediments more rapidly than 100S (100S+ DNA) has been observed (23,29,38). The 100S+ DNA has also been referred to as flowers (23,29).…”

“…In addition to the 32S to lOOS DNA, a replicating (and therefore branched) DNA that appears collapsed when viewed by electron microscopy and that sediments more rapidly than 100S (100S+ DNA) has been observed (23,29,38). The 100S+ DNA has also been referred to as flowers (23,29). Some T7 concatemers are part of 100S+ DNA (43,44).…”

“…Profiles suggesting the presence of discrete-length concatemers (dimers, etc.) are sometimes but not always seen a.fter rate-zonal centrifugation in sucrose gradients, followed by fractionation of the gradient and assay for DNA (16,21,23,37,50). However, the accuracy of the assay and the resolution pf the sucrose gradient are not sufficient to reliably detect discretelength concatemers in the presence of other types of DNA (variable-length concatemers, for example).…”