In vivo demonstration of enhanced binding between β-clamp and DnaE of pol III bearing consensus i-CBM
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Among 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.
The current study was aimed to evaluate in vivo interaction between DnaE bearing the consensus i-CBM and β-clamp.
The 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.
The 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.
The 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.
KeywordsPolymerase III β-Clamp CBM DnaE
We would like to thank HEC Pakistan and University of Sindh, Jamshoro for funding. We pay special regards to Dr. Karen A. Bunting and Dr. Jody A. Winter for their guidance during this research work.
AAP designed the project. Cloning of various constructs, i.e., experimental work was done by AAP and BBP. Both the authors contributed in writing the manuscript. The manuscript was reviewed by Dr. Nimerta Kumari (Ph. D. University of Tubingen Germany).
The project was funded by HEC Pakistan and University of Sindh, Jamshoro, and executed in the School of Biology, Queens Medical Centre, University of Nottingham, Nottingham, NG7 2UH, UK.
Compliance with ethical standards
Conflict of interest
Authors, Atif A. Patoli and Bushra B. Patoli declare no conflict of interest in the current manuscript.
It is also declared herewith that the current research work does not involve any human participant or animal model.
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