REPLICATION OF THE R6K-GAMMA ORIGIN IN-VITRO - DEPENDENCE ON WT-PI AND HYPERACTIVE PI-S87N PROTEIN VARIANT

The pi protein of plasmid R6K is involved in control of replication. T he aim of this study was to use an in vitro replication system depende nt on an R6K-derived gamma origin of replication (yori) to compare rep lication characteristics of wt pi and a hyperactive variant of pi prot ein (pi S87N; Filutowicz et al., 1994b. Cooperative binding of initiat or protein to replication origin conferred by single amino acid substi tution. Nucleic Acids Res. 22, 4211-4215). The characteristics of in v itro replication from yori reported in this investigation are as follo ws: (i) pi S87N is considerably more active in comparison to wt pi. (i i) Replication proceeds through Cairns-type intermediates and the init iation site and directionality of the fork movement are similar in the presence of both proteins. (iii) Replication forks emanate unidirecti onally in the vicinity of the cluster of seven 22-bp direct repeats wi thin yori. (iv) Replication dependent on wt pi, but not pi S87N, is st imulated up to 1.5-fold by rifampicin. (C) 1997 Elsevier Science B.V.