REPLICATION OF R6K GAMMA-ORIGIN IN-VITRO - DISCRETE START SITES FOR DNA-SYNTHESIS DEPENDENT ON PI AND ITS COPY-UP VARIANTS

The regulation of the plasmid R6K gamma origin (gamma ori) is accompli shed through the ability of the pi protein to act as an initiator and inhibitor of replication. Hyperactive variants of this protein, called copy-up pi, allow four to tenfold increases of gamma ori plasmid DNA in vivo. The higher activity of copy-up pi variants could be explained by an increase in the initiator function, a decrease in the inhibitor activity, or a derepression of a more efficient mechanism of replicat ion that can be used by wt pi (pi(35.0)) only under certain conditions . We have compared the replication activities of wt pi 35.0 and copy-u p pi mutants in vitro, and analyzed the replication products. It is sh own that copy-up variants are several-fold more active than wt pi(35.0 ) in replication. This appears to be due to enhanced specific replicat ion activity of copy-up mutants rather than elevated fractions of prot ein proficient in DNA binding. Furthermore, biochemical complementatio n revealed that pi 200 (copy-up) is dominant over wt pi(35.0). The ele vated activity of copy-up pi is not caused by an increased rate of rep lisome assembly as inferred from in vitro replication assays in which the lag periods observed were similar to that of wt pi(35.0). Moreover , only one round of semiconservative, unidirectional replication occur red in all the samples analyzed indicating that copy-up pi proteins do not initiate multiple rounds of DNA synthesis. Rather, a larger fract ion of DNA template replicates in the presence of copy-up pi as determ ined by electron microscopy. Two clusters of discrete DNA synthesis st art sites are mapped by primer extension near the stability (stb) locu s of the gamma ori. We show that the start sites are the same in the p resence of wt pi 35.0 or copy-up proteins. This comparative analysis s uggests that wt pi(35.0) and copy-up variants utilize fundamentally si milar mechanism(s) of replication priming. (C) 1998 Academic Press.