Reconstitution of R6K DNA replication in vitro using 22 purified proteins

Abstract

We have reconstituted a multiprotein system consisting of 22 purified proteins that catalyzed the initiation of replication specifically at ori gamma of R6K, elongation of the forks, and their termination at specific replication terminators. The initiation was strictly dependent on the plasmid-encoded initiator protein pi and on the host-encoded initiator DnaA. The wild type pi was almost inert, whereas a mutant form containing 3 amino acid substitutions that tended to monomerize the protein was effective in initiating replication. The replication in vitro was primed by DnaG primase, whereas in a crude extract system that had not been fractionated, it was dependent on RNA polymerase. The DNA-bending protein IHF was needed for optimal replication and its substitution by HU, unlike in the oriC system, was less effective in promoting optimal replication. In contrast, wild type pi-mediated replication in vivo requires IHF. Using a template that contained ori gamma flanked by two asymmetrically placed Ter sites in the blocking orientation, replication proceeded in the Cairns type mode and generated the expected types of termination products. A majority of the molecules progressed counterclockwise from the ori, in the same direction that has been observed in vivo. Many features of replication in the reconstituted system appeared to mimic those of in vivo replication. The system developed here is an important milestone in continuing biochemical analysis of this interesting replicon.