DNA binding properties of purified replication initiator protein (Rep) encoded by plasmid pSC101

We have purified the replication initiator protein (Rep) coded by plasmid pSC101. The purified protein was confirmed to be Rep by its amino-terminal sequence. Rep exists as a dimer and has a sequence-specific DNA-binding property. Protection experiments with DNA against cleavage by DNase I or exonuclease III showed that Rep bound preferentially to two nearly dyad-symmetric sequences overlapping the promoter of the rep gene, a structure gene of Rep. Transcripts in vitro from the rep promoter were identified and the precise initiation sites were determined by the primer-extension method. Rep represses the transcription from the rep promoter but not that from the bla gene promoter in the same reaction mixture, that is the rep gene is autoregulated. The replication origin (ori) of the plasmid contains directly repeated sequences similar to the symmetric sequences. However, a one order of magnitude higher concentration of the protein is required to bind to the origin repeats.