Twenty years of the pPS10 replicon: Insights on the molecular mechanism for the activation of DNA replication in iteron-containing bacterial plasmids

This review focuses on the contributions of the Pseudomonas replicon pPS10 to understanding the initiation of DNA replication in iteron-containing plasmids from Gram-negative bacteria. Dimers of the pPS10 initiator protein (RepA) repress repA transcription by binding to the two halves of an inverted repeat operator. RepA monomers are the active initiator species that bind to four directly repeated sequences (iterons). pPS10 initiator was the first Rep protein whose domains were defined (two “winged-helix,” WH modules) and their binding sites were identified at each half of the iteron repeat. This was confirmed by the crystal structure of the monomer of a homologous initiator (RepE from F plasmid) bound to iteron DNA. The recently solved structure of the dimeric N-terminal domain (WH1) of pPS10 RepA, when compared to the RepE monomer, shows that upon dimer dissociation an α-helix at WH1 C-terminus becomes part of an interdomain β-sheet. In solution, the iteron sequence, by itself, can induce the same kind of structural transformation in RepA. This seems to alter the package of both WH domains to adapt their DNA reading heads (HTH motifs) to the distinct spacing between half repeats in iterons and operator. Based on biochemical and spectroscopic work, structural and functional similarities were proposed between RepA and archaeal/eukaryal initiators. This was independently confirmed by the crystal structure of the archaeal initiator Cdc6. Characterization of mutants, either in pPS10 or in the Escherichia coli chromosome, has provided some evidence on a WH1-mediated interaction between RepA and the chromosomal initiator DnaA that results in a broadened-host range.Work on pPS10 has been possible thanks to the continuous support from Spanish CICyT/MCyT, currently under Grant BMC2003-00088. Finally, we are indebted to the enlightening work of many plasmid labs overseas, notably those focused in P1, F, R6K, RK2, and pSC101 replicons