The large staphylococcal multiresistance plasmids harbored in Gram-positive pathogens contribute significantly to the spread of multidrug-resistant bacteria and are typified by the presence of a highly conserved replication initiator protein, RepA, which is required for plasmid retention. RepA proteins contain N-terminal (NTD) and C-terminal (CTD) domains, which are both required for replication. We show that the RepA NTD and CTD show striking homology to the host primosome protein DnaD yet perform distinct functions; the NTD binds origin DNA in a novel manner and the CTD recruits the replicative helicase. Moreover, NTD–DNA structures reveal the first mechanism of origin handcuffing. Combined, the data unveil the minimal mechanism by which multiresistance plasmids mediate origin assembly via the highly conserved RepA protein.

Abstract

The staphylococcal multiresistance plasmids are key contributors to the alarming rise in bacterial multidrug resistance. A conserved replication initiator, RepA, encoded on these plasmids is essential for their propagation. RepA proteins consist of flexibly linked N-terminal (NTD) and C-terminal (CTD) domains. Despite their essential role in replication, the molecular basis for RepA function is unknown. Here we describe a complete structural and functional dissection of RepA proteins. Unexpectedly, both the RepA NTD and CTD show similarity to the corresponding domains of the bacterial primosome protein, DnaD. Although the RepA and DnaD NTD both contain winged helix-turn-helices, the DnaD NTD self-assembles into large scaffolds whereas the tetrameric RepA NTD binds DNA iterons using a newly described DNA binding mode. Strikingly, structural and atomic force microscopy data reveal that the NTD tetramer mediates DNA bridging, suggesting a molecular mechanism for origin handcuffing. Finally, data show that the RepA CTD interacts with the host DnaG primase, which binds the replicative helicase. Thus, these combined data reveal the molecular mechanism by which RepA mediates the specific replicon assembly of staphylococcal multiresistant plasmids.