Structural Changes in RepA, a plasmid replication initiator, upon binding to origin DNA

Abstract

RepA protein is the DNA replication initiator of the Pseudomonas plasmid pPS10.RepA dimers bind to an inversely-repeated operator sequence in repA promoter, thus repressing its own synthesis, whereas monomers bind to four directly-repeated sequences(iterons) to initiate DNA replication. We had previously proposed that RepA is composed of two Winged-Helix (WH) domains, a structural unit also present in eukaryotic and archaeal initiators. In order to bind to the whole iteron sequence through both domains, RepA should couple monomerization to a conformational change in the N-terminal WH, which includes a Leucine-Zipper (LZ)-like sequence motif. We show for the first time that, by itself, binding to iteron DNA in vitro: i) dissociates RepA dimers into monomers and ii) alters RepA conformation, suggesting an allosteric effect. Furthermore, we also show that similar changes in RepA are promoted by mutations that substitute two Leu residues of the putative LZ by Ala, destabilizing the hydrophobic core of the first WH. We propose that this mutant (RepA- 2L2A) resembles a transient folding intermediate in the pathway leading to active monomers.These findings, together with the known activation of other Rep-type proteins by chaperones,are relevant to understand the molecular basis of plasmid DNA replication initiation.