In vitro synthesis of bacteriophage phi X174 by purified components.

Abstract

An in vitro system capable of synthesizing infectious 4X174 phage particles was reconstituted from purified components. The synthesis required 4X174 supercoiled replicative form DNA, OX174-encoded proteins A, C, J, and prohead, Esch-erichia coli DNA polymerase III holoenzyme, rep protein, and deoxyuridinetriphosphatase (dUTPase, dUTP nucleotidohydrolase, EC 3.6.1.23) as well as MgCl2, four deoxyribonucleoside triphosphates, and ATP. Phage production was coupled to the synthesis of viral single-stranded DNA. More than 70% of the synthesized particles sedimented at the position of mature phage in a sucrose gradient and associated with the infectivity. The simple requirement of the host proteins suggests that the mechanism of viral strand synthesis in the phage-synthesizing reaction resembles that of viral strand synthesis during the replication of replicative form DNA. (11,12) to form the replication assembly sedimenting at 50S when centrifuged in sucrose gradients (50S complex) (13). Gene C protein is thought to be required for association of the prohead and the template (14). Stage III DNA synthesis proceeds in the 50S complex by a rolling circle mechanism (13, 15) with the displaced viral strand packaged into the prohead. Gene J protein may be incorporated into phage particles during these processes. After one round of replication is completed, the A protein cleaves the viral strand at ori(+) and joins the two ends to form a circular genome packaged in the phage particle (16).To study this whole process in detail, we have been developing in vitro systems capable of stage III DNA synthesis leading to the formation of mature phage (12, 17). Previouslv, we described an in vitro system composed of purified phage-encoded protein components and an unfractionated E. coli protein fraction (18). In this system, we found that phage synthesis requires 4X174 RFI DNA as template, 4X174-encoded proteins A, C, and J and prohead. Recently, we also showed that a fragment of RF DNA containing ori(+) carries 4195The publication costs of this article were defrayed in part by page charge payment. This article must therefore be herebv marked "advertisement" in accordance with 18 U.S.C. §1734 solelv to indicate this fact.

Mutational analysis of the bacteriophage φX174 replication origin

“…In this stage rolling circle DNA replication is strictly coupled to phage morphogenesis. The last three nucleotides may be part of a morphogenetic signal, which may be required for the &X174 gene C protein-mediated attachment of the prohead to the DNA (Aoyama & Hayashi, 1986). The failure to obtain (6X174 mutants at these positions is in agreement with the results of these transduction studies.…”

“…and DNA packaging (Aoyama & Hayashi, 1986). In this way the displaced viral strand is replicated directly into the phage prohead (Koths & Dresiler, 1980;Aoyama et al, 1983).…”

DNA replication of single-stranded Escherichia coli DNA phages

“…Therefore, it is not available to enzymes which normally convert single-stranded circles into duplex rings earlier in the viral life cycle [78,114,115,125]. Stage III replication has been accomplished with purified components in vitro in the laboratory of Hayashi [126]. In this system the RFI templates undergo several rounds of DNA synthesis, the same replicative intermediates are observed as in vivo and viable phage particles are the end-products of the reaction.…”

“…These proteins are not necessary in the purified in vitro system [126]. It is possible that the DNA gyrase subunit A [129] conversion to or maintenance of stage III replication in the presence of other replication enzymes.…”

“…In this system the RFI templates undergo several rounds of DNA synthesis, the same replicative intermediates are observed as in vivo and viable phage particles are the end-products of the reaction. Chimeric plasmids containing a functional origin are packaged with the same efficiency as eX RFI, provided the length of the plasmid is approximately the same as that of eX DNA [103,126]. A pictorial representation of their model is shown in Fig.…”