Replication of phi X174 dna with purified enzymes. I. Conversion of viral DNA to a supercoiled, biologically active duplex.

Conversion of phi X174 viral, single-stranded circular DNA to the duplex replicative form (RF), previously observed with partially purified enzymes, has now been demonstrated with the participation of 12 nearly pure Escherichia coli proteins containing approximately 30 polypeptides. To complete the synthesis of a full length complementary strand, E. coli DNA polymerase I was needed to fill the short gap left by DNA polymerase III holoenzyme, and to remove the primer and replace it with DNA. Production of supercoiled RF required the further actions of E. coli DNA ligase and gyrase. Net synthesis of viral circles was obtained by coupling the formation of RF supercoils to the actions of the phi X174-encoded gene A protein and E. coli rep protein. Viral DNA circles produced from enzymatically synthesized supercoiled RF, serving as template-substrate, were indistinguishable from those produced from RF isolated from infected cells; synthetic RF and the viral circles generated from it by replication were as biologically active in transfection of spheroplasts as the forms obtained from infected cells and virions. The conversion of single-stranded circular DNA to RF is suggested here as a model for discontinuous synthesis of the lagging strand of the E. coli chromosome. The primosome, a complex of some of the replication proteins responsible for initiations of DNA chains, will be described elsewhere. Multiplication of RF supercoils, described in the succeeding paper, proceeds by a rolling-circle mechanism in which the synthesis of viral strands may have analogies to the continuous synthesis of the leading strand of the E. coli chromosome.

The 2.2 kb E1b mRNA of human Ad12 and Ad5 codes for two tumor antigens starting at different AUG triplets.

By nucleotide sequence analysis and S1 nuclease mapping we have determined the structural organization of early region E1b of Ad12. We have also revised the nucleotide sequence of the E1b region of Ad5. Both regions have an identical structural organization and show considerable homology at the nucleotide level. The major tumor antigens (Ad12, 19 and 54 kilodaltons [kd]); Ad5, 21 and 55 kd) are encoded in two overlapping reading frames. A single mRNA of 2.2 kilobases codes for both these proteins, depending on which AUG triplet serves as the start codon: the 19-21 kd protein initiates at the 5'-promximal AUG; the 54-55 kd protein initiates at the second AUG in another reading frame. Peptide mapping shows that the small and large tumor antigens do not share common tryptic peptides, in accordance with the nucleic acid sequence data. In addition, the 19-21 kd protein can also by synthesized from a one kilobase mRNA. Finally, the gene for the Ad12 analog of protein IX is characterized.