Atif A Patoli1,2, Bushra B Patoli3,4. 1. School of Biology, Queens Medical Centre, University of Nottingham, Nottingham, NG72UH, UK. atifpatoli@gmail.com. 2. Institute of Microbiology, University of Sindh, Jamshoro, Pakistan. atifpatoli@gmail.com. 3. School of Biology, Queens Medical Centre, University of Nottingham, Nottingham, NG72UH, UK. 4. Institute of Microbiology, University of Sindh, Jamshoro, Pakistan.
Abstract
BACKGROUND: 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. OBJECTIVE: The current study was aimed to evaluate in vivo interaction between DnaE bearing the consensus i-CBM and β-clamp. METHOD: 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. RESULT: 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. CONCLUSION: 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.
BACKGROUND: 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. OBJECTIVE: The current study was aimed to evaluate in vivo interaction between DnaE bearing the consensus i-CBM and β-clamp. METHOD: 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. RESULT: 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. CONCLUSION: 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.
Authors: Roxana E Georgescu; Isabel Kurth; Nina Y Yao; Jelena Stewart; Olga Yurieva; Mike O'Donnell Journal: EMBO J Date: 2009-08-20 Impact factor: 11.598