Synthesis of Bacteriophage qXl74 In Vitro: Mechanism of Switch from to DNA Packaging DNA Replication

Akira Aoyama and Masaki Hayashi Department of Biology University of California at San Diego La Jolla, California 92093 Replication of a replicative form DNA of bacterio- phage 1~x174 initiates by rolling-circle synthesis of the viral DNA followed by discontinuous synthesis of the complementary DNA. Gene C protein of (pX174, which is involved in DNA packaging, inhibits the rolling- circle DNA synthesis by binding to the initiation com- plex in vitro. The gene C protein-associated initiation complex can synthesize and package the viral DNA to produce infectious phage when supplemented with (pX174 gene J protein and the prohead. Multiple rounds of phage synthesis occur without dissociation of the gene C protein from the complex. These results indi- cate that gene C protein is central in the switch from replication of a replicative form DNA to synthesis and concomitant packaging of viral DNA into phage cap- sid, which occurs in the late stage of infection. Introduction Bacteriophage (pX174 contains a circular single-stranded (SS) DNA in an icosahedral protein capsid. Packaging of the SS DNA occurs via three successive stages of DNA synthesis (Dressler et al., 1978; Hayashi, 1978). After in- fection of E. coli, the SS DNA is converted to a circular double-stranded (DS) replicative form (RF) DNA (SS-to- RF synthesis). The RF DNA, then, serves as template for semiconservative replication of RF DNA (RF-to-RF syn- thesis). After several rounds of replication, the RF DNA changes its role to serve as template for an asymmetric synthesis of the viral SS DNA. This process is tightly cou- pled to the packaging of the viral DNA in phage particles (RF-to-Phage synthesis). The mechanism of the switch- over of the role of the RF DNA is, therefore, important to understanding the mechanism of the DNA packaging pro- cess, which is one of the highlights of the morphogenesis of (px174. Replication of the RF DNA initiates by rolling-circle syn- thesis of the viral strand (RF-to-SS synthesis), followed by discontinuous synthesis of the complementary strand (Eisenberg et al., 1978; Arai et al., 1981). The mechanisms of these processes are well understood from in vitro ex- periments. The RF-to-SS DNA synthesis requires (pX174 gene A protein, E. coli DNA polymerase III holoenzyme, rep protein, and single-stranded DNA binding protein (SSB) (Scott et al., 1977). Gene A protein initiates the RF- to-SS DNA synthesis by introducing a nick on the super- coiled RF (RF I) DNA at the origin of DNA replication (Franke and Ray, 1972; Langeveld et al., 1978) and at- taches covalently to the 5’ end of the viral strand to form an open circular RF (RF II) DNA (RF II-A complex) (Eisen- berg and Kornberg, 1979). Rep protein binds to the RF II-A complex at the origin (RF II-A-rep complex) and unwinds the DNA strand, consuming the energy of ATP hydrolysis (Scott et al., 1977; Kornberg et al., 1978; Yarranton and Gefter, 1979; Arai and Kornberg, 1981). Strand separation is maintained by SSB, which binds to the separated DNA strands. DNA polymerase III holoenzyme catalyzes the rolling-circle synthesis of the viral DNA by extending the 3’ end of the viral DNA, while the 5’ end of the viral strand is being displaced. After one round of the rolling-circle synthesis, the gene A protein cuts the viral strand at the origin and rejoins the two ends to generate the circular SS DNA covered with SSB (Eisenberg et al., 1977). The gene A protein is transferred to the newly generated 5’ end of the RF DNA and forms II-A-rep complex, which serves as template for the next round of RF-to-SS synthe- sis (Eisenberg and Kornberg, 1979; Brown et al., 1984). The discontinuous synthesis of the complementary strand starts during or after the RF-to-SS synthesis when the SSB-coated viral strand is available for the primosome to initiate RNA priming, which generates RF I DNA (Arai et al., 1981). The asymmetric synthesis of viral DNA and its concomi- tant packaging is initiated by the nicking action of gene A protein on RF I DNA and proceeds via a rolling-circle mode similar to that described for the RF replication (Aoyama et al., 1983a). The process requires @X174 gene C protein (a nonstructural single-stranded specific DNA binding protein; Aoyama et al., 1983b), gene J protein (a highly basic structural protein of phage; Freymeyer et al., 1977) and prohead (a phage head precursor made of gene F, G, H, B, and D proteins; Fujisawa and Hayashi, 1977b; Mukai et al., 1979) in addition to the components required for rolling-circle DNA synthesis in the RF replication (Aoyama et al., 1983a). SSB is not essential in the in vitro RF-to-Phage system (Aoyama et al., 1983a). Synthesis of the viral DNA occurs at the replication fork in RF DNA that is associated with the prohead in which the displaced viral strand is directly packaged during the rolling-circle DNA synthesis (Fujisawa and Hayashi, 1977b; Koths and Dressler, 1980; Aoyama et al., 1983a). Gene C protein is thought to be required for the association of prohead to the template DNA (Aoyama et al., 1983a). After one round of DNA replication, a circular viral DNA is formed within the prohead by the cutting-rejoining activity of the gene A protein (Fujisawa and Hayashi, 1978). The products of this reaction are infectious phage particle and the RF II DNA template, which is used for subsequent rounds of phage synthesis. A possible mechanism of the conversion from RF-to-RF mode to RF-to-Phage mode had been previously sug- gested from the in vivo experiments. Fujisawa and Ha- yashi observed that the newly synthesized open struc- tural RF DNA is rapidly converted to I DNA, while the prohead structure accumulates in cells that are infected with gene C mutants (Fujisawa and Hayashi, 1977a). The