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KMID : 0381120190410060613
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Genes and Genomics
2019 Volume.41 No. 6 p.613 ~ p.619
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In vivo demonstration of enhanced binding between β-clamp and DnaE of pol III bearing consensus i-CBM
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Patoli Atif A.
Patoli Bushra B.
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Abstract
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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.
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KEYWORD
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Polymerase III, β-Clamp, CBM, DnaE
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