FULLY METHYLATED ORIC WITH NEGATIVE SUPERHELICITY FORMS AN ORIC-MEMBRANE COMPLEX BEFORE INITIATION OF CHROMOSOME-REPLICATION

In an in vitro assay, the oriC DNA has been shown to bind to the outer membrane fraction only when it is hemimethylated (G.B. Ogden et al., Cell, 54, 127-135, 1988). In this report, however, we demonstrated that a significant amount of the oriC DNA was recovered from the cells just before initiation with the oriC DNA being fully methylated. Formation of this preinitiation oriC-membrane complex and following initiation of chromosome replication were strongly inhibited by novobiocin, a DNA gyrase B subunit inhibitor, which reduced the superhelicity of the reporter plasmid in the cells. On the other hand, both reactions proceeded in the presence of nalidixic acid, a DNA gyrase A subunit inhibitor, which did not have the effect of reducing the superhelicity. These results suggest that the negative superhelicity of the DNA is required for preinitiation oriC-membrane complex formation and following initiation event of replication. © 1993 Academic Press, Inc.