A pseudo-symmetric DNA binding motif of purified Rep dimer of plasmid pSC101

The Rep protein, a replication initiator of plasmid pSC101, also functions as an autorepressor for its own structure gene, rep, through binding to the operator which consists of imperfectly dyad-symmetric (pseudo-symmetric) sequences, IR-1 and IR-2. In order to define the DNA binding motif of Rep, we have analyzed its binding affinity for each half repeat of the IR sequences and found that the right half of IR-2 was the preferred sequence for Rep, although its affinity was much lower than those of the IR sequences. Next, dimeric sequences of each half repeat with different configurations, head to head (the same configuration as natural IRs), head to tail, and tail to tail, were constructed and their affinities for Rep were examined. Almost all of sequences with the head-to-head configuration had binding affinity for Rep but these, including even the symmetric sequence of the right half of IR-2, exhibited lower Rep-binding abilities than natural IR sequences. We presume a pseudo-symmetric sequence, 5'GGNNTAGNNATTNNNATNN(N)CTAGNCC3', to be the Rep-binding motif from a sequence comparison. Some single-base substitution experiments confirmed the motif and suggested that each subunit of the Rep dimer recognizes each half repeat of IR asymmetrically.