Investigations of pi initiator protein-mediated interaction between replication origins alpha and gamma of the plasmid R6K.

A typical plasmid replicon of Escherichia coli, such as ori gamma of R6K, contains tandem iterons (iterated initiator protein binding sites), an AT-rich region that melts upon initiator-iteron interaction, two binding sites for the bacterial initiator protein DnaA, and a binding site for the DNA-bending protein IHF. R6K also contains two structurally atypical origins called alpha and beta that are located on either side of gamma and contain a single and a half-iteron, respectively. Individually, these sites do not bind to initiator protein pi but access it by DNA looping-mediated interaction with the seven pi-bound gamma iterons. The pi protein exists in 2 interconvertible forms: inert dimers and active monomers. Initiator dimers generally function as negative regulators of replication by promoting iteron pairing ("handcuffing") between pairs of replicons that turn off both origins. Contrary to this existing paradigm, here we show that both the dimeric and the monomeric pi are necessary for ori alpha-driven plasmid maintenance. Furthermore, efficient looping interaction between alpha and gamma or between 2 gamma iterons in vitro also required both forms of pi. Why does alpha-gamma iteron pairing promote alpha activation rather than repression? We show that a weak, transitory alpha-gamma interaction at the iteron pairs was essential for alpha-driven plasmid maintenance. Swapping the alpha iteron with one of gamma without changing the original sequence context that caused enhanced looping in vitro caused a significant inhibition of alpha-mediated plasmid maintenance. Therefore, the affinity of alpha iteron for pi-bound gamma and not the sequence context determined whether the origin was activated or repressed.