Convergent evolution involving dimeric and trimeric dUTPases in pathogenicity island mobilization
(A) Superimposition of three-dimensional structures of the ϕDI dimer in apo form (yellow tones), dUPNPP bound (magenta tones) and as A73L mutant (green tones) in cartoon representation. The nucleotide in the ϕDI-dUPNPP structure is represented in stick with carbon atoms in cyan and Mg as green spheres. (B) SaPIbov1 excision and replication following induction of the cloned ϕDIA73L dut gene. Strain JP6774 containing SaPIbov1 was complemented with plasmids expressing the 3xFLAG-tagged ϕDI and ϕDIA73L dimeric Dut or the empty pCN51 plasmid as a control. Samples were isolated at 3 hours after induction with 3μM CdCl2 and Southern blots were performed using a probe for the SaPIbov1 integrase (S4 Table). The upper band is ‘bulk’ DNA, including chromosomal, phage, and replicating SaPI. CCC indicates covalently closed circular SaPI DNA. In these experiments, as no helper phage was present, the excised and replicating SaPI DNA appears as part of the bulk DNA or as CCC molecules, rather than the linear monomers that are seen following helper phage-mediated induction and packaging. (C) Native-PAGE experiment was performed to analyze the binding capacity of the ϕDIA73L Dut vs the ϕDI wild-type form. Proteins were mixed with Stl in equimolecular relationship (in monomers). A clear reduction in the band corresponding to the Stl-ϕDI DutA73L complex with respect to the Stl-ϕDI Dut complex is observed. (D) Close-up view of the superimposed ϕDI-dUPNPP and ϕDIA73L active centers. Residues responsible for substrate coordination and binding are represented in sticks with carbon atoms colored according to the structure to which they correspond. The Leu73 residue at the bottom of the active center of the ϕDIA73L mutant that precludes the binding of the nucleotide is colored in yellow. The substrate dUPNPP in the ϕDI-dUPNPP structure is represented in stick with carbon atoms in cyan and Mg ions are represented as grey spheres.