Mignan, Sirine El Houry; Witte, Gregor; Naue, Natalie; Curth, Ute: Characterization of the chi psi subcomplex of Pseudomonas aeruginosa DNA polymerase III. In: BMC Molecular Biology 2011, 12:43




Background: DNA polymerase III, the main enzyme responsible for bacterial DNA replication, is composed of three sub-assemblies: the polymerase core, the beta-sliding clamp, and the clamp loader. During replication, single-stranded DNA-binding protein (SSB) coats and protects single-stranded DNA (ssDNA) and also interacts with the chi psi heterodimer, a sub-complex of the clamp loader. Whereas the chi subunits of Escherichia coli and Pseudomonas aeruginosa are about 40% homologous, P. aeruginosa psi is twice as large as its E. coli counterpart, and contains additional sequences. It was shown that P. aeruginosa chi psi together with SSB increases the activity of its cognate clamp loader 25-fold at low salt. The E. coli clamp loader, however, is insensitive to the addition of its cognate chi psi under similar conditions. In order to find out distinguishing properties within P. aeruginosa chi psi which account for this higher stimulatory effect, we characterized P. aeruginosa chi psi by a detailed structural and functional comparison with its E. coli counterpart. Results: Using small-angle X-ray scattering, analytical ultracentrifugation, and homology-based modeling, we found the N-terminus of P. aeruginosa psi to be unstructured. Under high salt conditions, the affinity of the chi psi complexes from both organisms to their cognate SSB was similar. Under low salt conditions, P. aeruginosa chi psi, contrary to E. coli chi psi, binds to ssDNA via the N-terminus of psi. Whereas it is also able to bind to double-stranded DNA, the affinity is somewhat reduced. Conclusions: The binding to DNA, otherwise never reported for any other psi protein, enhances the affinity of P. aeruginosa chi psi towards the SSB/ssDNA complex and very likely contributes to the higher stimulatory effect of P. aeruginosa chi psi on the clamp loader. We also observed DNA-binding activity for P. putida chi psi, making this activity most probably a characteristic of the psi proteins from the Pseudomonadaceae.