UNCOUPLING OF THE DNA TOPOISOMERASE AND REPLICATION ACTIVITIES OF AN INITIATOR PROTEIN

The replication initiator proteins encoded by the pT181 and related pl asmids have sequence-specific DNA binding and topoisomerase activities . These proteins create a site-specific nick in one strand of the DNA at the origin of replication that serves as a primer for the initiatio n of replication. To define the regions of the pT181-encoded initiator protein, RepC, that are involved in its DNA binding, topoisomerase, a nd replication activities, we have carried out site-directed mutagenes is of the repC gene. Analysis of mutant RepC proteins in vitro and in vivo has identified the amino acids that are critical for its various biochemical activities. The DNA binding domain of RepC was found to be located near its C-terminal region and was different from the domain involved in its sequence-specific topoisomerase activity. These studie s also showed that the DNA topoisomerase activity of the initiator pro tein can be uncoupled from its tight noncovalent DNA binding and repli cation activities.