ArticleGenes & genomics2019
In vivo demonstration of enhanced binding between β-clamp and DnaE of pol III bearing consensus i-CBM.
Article in Genes & genomics, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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2 authors at 1 institution in 2 countries.
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Abstract
backgroundAmong several key protein-protein and protein-DNA interactions within the replisome, the interaction between β-clamp and the DNA polymerase (Pol) III is of crucial importance. This interaction is mediated by a five or six-residue conserved sequence of the DnaE subunit of Pol III, referred to as the Clamp Binding Motif (CBM). In E. coli, DnaE contains two CBMs designated as e-CBM and i-CBM. A consensus sequence (QL[S/D]LF) for the CBMs has previously been proposed and studies involving mutagenesis of both the CBMs have evaluated their protein-binding properties. Surface Plasmon Resonance has been used to show that replacing i-CBM in DnaE with the consensus sequence enhances its binding to β-clamp 120-fold.
objectiveThe current study was aimed to evaluate in vivo interaction between DnaE bearing the consensus i-CBM and β-clamp.
methodThe C-terminal 405 residues of DnaE, bearing either the consensus i-CBM or the WT i-CBM, with β-clamp were co-expressed in E. coli followed by co-purification of the protein complexes. The interaction was assessed by the ability of the co-expressed proteins to form stable complexes during both affinity and gel filtration chromatography.
resultThe interaction of β-clamp with DnaEΔ755M containing the consensus i-CBM was found to be more stable than with WT DnaEΔ755, consistent with the in vitro data previously reported.
conclusionThe presence of the pieces of sheared DNA generated during sonication promote the interaction of DnaEΔ755M with β-clamp by binding the OB-fold of DnaEΔ755M and β-clamp and serves as a bridge between them.
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