INITIATOR PROTEIN-PI CAN BIND INDEPENDENTLY TO 2 DOMAINS OF THE GAMMA-ORIGIN CORE OF PLASMID R6K - THE DIRECT REPEATS AND THE A-RICH SEGMENT(T)

The pi protein of plasmid R6K functions in both replication and transc ription. pi autoregulates its own synthesis and is required for replic ation of the R6K gamma origin. pi performs these functions by binding to specific DNA sites arranged as pairs of 6-10 bp inverted repeats (I Rs) or as a cluster of seven tandem 22 bp direct repeats (DRs) which l ack 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 gam ma origin. In this work we carried out DNase I and hydroxyl radical pr otection experiments on various deletion derivatives of the gamma orig in complexed with pi protein. These experiments revealed binding of pi to a novel site embedded within the A+T-rich segment. This interactio n manifests primarily by the appearance of the enhanced scissions of D NA 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 gam ma origin.