Negative regulation of pPS10 plasmid replication: origin pairing by zipping-up DNA-bound RepA monomers

In many plasmid replicons of gram-negative bacteria, Rep protein dimers are transcriptional self-repressors of their genes, whereas monomers are initiators of DNA replication. Switching between both functions implies conformational remodelling of Rep, and is promoted by Rep binding to the origin DNA repeats (iterons) or chaperones. Rep proteins play another key role: they bridge together two iteron DNA stretches, found either on the same or on different plasmid molecules. These so-called, respectively, 'looped' and 'handcuffed' complexes are thought to be negative regulators of plasmid replication. Although evidence for Rep-dependent plasmid handcuffing has been found in a number of replicons, the structure of these Rep-DNA assemblies is still unknown. Here, by a combination of proteomics, electron microscopy, genetic analysis and modelling, we provide insight on a possible three-dimensional structure for two handcuffed arrays of the iterons found at the origin of pPS10 replicon. These are brought together in parallel register by zipping-up DNA-bound RepA monomers. We also present evidence for a distinct role of RepA dimers in DNA looping. This work defines a new regulatory interface in Rep proteins.