Initiator protein pi can bind independently to two domains of the gamma origin core of plasmid R6K: the direct repeats and the A+T-rich segment.

The pi protein of plasmid R6K functions in both replication and transcription. pi autoregulates its own synthesis and is required for replication of the RISK gamma origin. pi performs these functions by binding to specific DNA sites arranged as pairs of 6-10 bp inverted repeats (IRs) or as a cluster of seven tandem 22 bp direct repeats (DRs) which lack symmetry. The sites share the TGAGRG nucleotide motif (where R is A or G). The DRs and IRs flank the central A+T-rich segment of the gamma origin. In this work we carried out DNase I and hydroxyl radical protection experiments on various deletion derivatives of the gamma origin complexed with pi protein. These experiments revealed binding of pi to a novel site embedded within the A+T-rich segment. This interaction manifests primarily by the appearance of the enhanced scissions of DNA by DNase I and hydroxyl radicals. pi interaction with the A+T-rich site is independent of pi binding to the DRs and IRs. We propose that pi protein can recognize distinct families of DNA sequences in the gamma origin.