Plasmid RK2 ParB protein: Purification and nuclease properties

The parCBA operon of the 3.2-kb stabilization region of plasmid RK2 encodes three cotranslated proteins. ParA mediates site-specific recombination to resolve plasmid multimers, ParB has been shown to be a nuclease, and the fu nction of ParC is unknown. In this study ParB was overexpressed by cotransl ation with ParC in Escherichia coli by using a plasmid construct that conta ined the parC and parB genes under the control of the T7 promoter. Purifica tion was achieved by treatment of extracts with Polymin P, followed by ammo nium sulfate precipitation and heparin and ion-exchange chromatography. Siz ing-column analysis indicated that ParB exists as a monomer in solution. An alysis of the enzymatic properties of purified ParB indicated that the prot ein preferentially cleaves single-stranded DNA. ParB also nicks supercoiled plasmid DNA preferably at sites with potential single-stranded character, like AT-rich regions and sequences that can form cruciform structures, ParB also exhibits 5'-->3' exonuclease activity. This ParB activity on a 5'-end -labeled, double-stranded DNA substrate produces a 3',5'-phosphorylated din ucleotide which is further cleaved to a 3',5'-phosphorylated mononucleotide . The role of the ParB endonuclease and exonuclease activities in plasmid R K2 stabilization remains to be determined.