Replication of duplex DNA of phage φX174 reconstituted with purified enzymes

Replication of the covalently closed duplex replicative form (RF) of phage ∅X174 DNA has been achieved by coupling two known enzyme systems: (i) synthesis of viral strand circles (SS) from RF, and (ii) conversion of SS to nearly complete RF (RF II). In this coupled system, activated RF (gene A·RF II complex) was a more efficient template and generated as many as 10 RF II molecules per RF input, at a rate commensurate with SS synthesis. The 11 proteins required for the two component systems were all needed in the coupled RF duplication system; no new factors were required. Single-stranded DNA binding protein was needed for RF duplication at only 4% the level needed in its stoichiometric participation in SS synthesis. In addition to RF II, more complex replicative forms appeared late in the reaction, and their possible origin is discussed.