Origin pairing (‘handcuffing’) and unpairing in the control of P1 plasmid replication

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Abstract
Summary: The P1 plasmid origin has an array of five binding sites (iterons) for the plasmid‐encoded initiator protein RepA. Saturation of these sites is required for initiation. Iterons can also pair via their bound RepAs. The reaction, called handcuffing, is believed to be the key to control initiation negatively. Here we have determined some of the mechanistic details of the reaction. We show that handcuffed RepA–iteron complexes dissociate when they are diluted or challenged with cold competitor iterons, suggesting spontaneous reversibility of the handcuffing reaction. The complex formation increases with increased RepA binding, but decreases upon saturation of binding. Complex formation also decreases in the presence of molecular chaperones (DnaK and DnaJ) that convert RepA dimers to monomers. This indicates that dimers participate in handcuffing, and that chaperones are involved in reversing handcuffing. They could play a direct role by reducing dimers and an indirect role by increasing monomers that would compete out the weaker binding dimers from the origin. We propose that an increased monomer to dimer ratio is the key to reverse handcuffing.