Crystal structure of the chi:psi subassembly of the Escherichia coli DNA polymerase clamp-loader complex

Abstract

The chi (χ) and psi (ψ) subunits of Escherichia coli DNA polymerase III form a heterodimer that is associated with the ATP-dependent clamp-loader machinery. In E. coli, the χ:ψ heterodimer serves as a bridge between the clamp-loader complex and the single-stranded DNA-binding protein. We determined the crystal structure of the χ:ψ heterodimer at 2.1 Å resolution. Although neither χ (147 residues) nor ψ (137 residues) bind to nucleotides, the fold of each protein is similar to the folds of mononucleotide-(χ) or dinucleotide-(ψ) binding proteins, without marked similarity to the structures of the clamp-loader subunits. Genes encoding χ and ψ proteins are found to be readily identifiable in several bacterial genomes and sequence alignments showed that residues at the χ:ψ interface are highly conserved in both proteins, suggesting that the heterodimeric interaction is of functional significance. The conservation of surface-exposed residues is restricted to the interfacial region and to just two other regions in the χ:ψ complex. One of the conserved regions was found to be located on χ, distal to the ψ interaction region, and we identified this as the binding site for a C-terminal segment of the single-stranded DNA-binding protein. The other region of sequence conservation is localized to an N-terminal segment of ψ (26 residues) that is disordered in the crystal structure. We speculate that ψ is linked to the clamp-loader complex by this flexible, but conserved, N-terminal segment, and that the χ:ψ unit is linked to the single-stranded DNA-binding protein via the distal surface of χ. The base of the clamp-loader complex has an open C-shaped structure, and the shape of the χ:ψ complex is suggestive of a loose docking within the crevice formed by the open faces of the δ and δ′ subunits of the clamp-loader.