Replication of the R6K gamma origin in vitro: dependence on wt pi and hyperactive piS87N protein variant.

The pi protein of plasmid R6K is involved in control of replication. The aim of this study was to use an in vitro replication system dependent on an R6K-derived gamma origin of replication (gamma ori) to compare replication characteristics of wt pi and a hyperactive variant of pi protein (piS87N; Filutowicz et al., 1994b. Cooperative binding of initiator protein to replication origin conferred by single amino acid substitution. Nucleic Acids Res. 22, 4211-4215). The characteristics of in vitro replication from gamma ori reported in this investigation are as follows: (i) piS87N is considerably more active in comparison to wt pi. (ii) Replication proceeds through Cairns-type intermediates and the initiation site and directionality of the fork movement are similar in the presence of both proteins. (iii) Replication forks emanate unidirectionally in the vicinity of the cluster of seven 22-bp direct repeats within gamma ori. (iv) Replication dependent on wt pi, but not piS87N, is stimulated up to 1.5-fold by rifampicin.