Advertisement

In vivo demonstration of enhanced binding between β-clamp and DnaE of pol III bearing consensus i-CBM

  • Atif A. PatoliEmail author
  • Bushra B. Patoli
Research Article
  • 20 Downloads

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.

Keywords

Polymerase III β-Clamp CBM DnaE 

Notes

Acknowledgements

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.

Author contributions

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).

Funding

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.

Ethical approval

It is also declared herewith that the current research work does not involve any human participant or animal model.

References

  1. Burnouf DY, Olieric V, Wagner J, Fujii S, Reinbolt J, Fuchs RPP, Dumas P (2004) Structural and biochemical analysis of sliding clamp/ligand interactions suggest a competition between replicative and translesion DNA polymerases. J Mol Biol 335:1187–1197.  https://doi.org/10.1016/j.jmb.2003.11.049 CrossRefGoogle Scholar
  2. Dalrymple BP, Kongsuwan K, Wijffels G, Dixon NE, Jennings PA (2001) A universal protein–protein interaction motif in the eubacterial DNA replication and repair systems. Proc Natl Acad Sci 98:11627–11632.  https://doi.org/10.1073/pnas.191384398 CrossRefGoogle Scholar
  3. Deininger PL (1983) Random subcloning of sonicated DNA: application to shotgun DNA sequence analysis. Anal Biochem 129:216–223.  https://doi.org/10.1016/0003-2697(83)90072-6 CrossRefGoogle Scholar
  4. Doherty AJ, Serpell LC, Ponting CP (1996) The helix-hairpin-helix DNA-binding motif: a structural basis for non-sequence-specific recognition of DNA. Nucleic Acids Res 24:2488–2497.  https://doi.org/10.1093/nar/24.13.2488 CrossRefGoogle Scholar
  5. Dohrmann PR, McHenry CS (2005) A bipartite polymerase-processivity factor interaction: only the internal β binding site of the α subunit is required for processive replication by the DNA polymerase III holoenzyme. J Mol Biol 350:228–239.  https://doi.org/10.1016/j.jmb.2005.04.065 CrossRefGoogle Scholar
  6. Duzen JM, Walker GC, Sutton MD (2004) Identification of specific amino acid residues in the E. coli β processivity clamp involved in interactions with DNA polymerase III, UmuD and UmuD′. DNA Repair 3:301–312.  https://doi.org/10.1016/j.dnarep.2003.11.008 CrossRefGoogle Scholar
  7. Fribourg S, Romier C, Werten S, Gangloff YG, Poterszman A, Moras D (2001) Dissecting the interaction network of multiprotein complexes by pairwise coexpression of subunits in E. coli. J Mol Biol 306:363–373.  https://doi.org/10.1006/jmbi.2000.4376 CrossRefGoogle Scholar
  8. Georgescu RE, Kurth I, Yao NY, Stewart J, Yurieva O, O’Donnell M (2009) Mechanism of polymerase collision release from sliding clamps on the lagging strand. EMBO J 28:2981–2991.  https://doi.org/10.1038/emboj.2009.233 CrossRefGoogle Scholar
  9. Johnson A, O’Donnell M (2005) Cellular DNA replicases: components and dynamics at the replication fork. Annu Rev Biochem 74:283–315.  https://doi.org/10.1146/annurev.biochem.73.011303.073859 CrossRefGoogle Scholar
  10. Kuriyan J, O’Donnell M, Yurieva O, Georgescu RE, Kim S-S, Kong X-P (2008) Structure of a sliding clamp on DNA. Cell 132:43–54.  https://doi.org/10.1016/j.cell.2007.11.045 CrossRefGoogle Scholar
  11. Lamers MH, Georgescu RE, Lee SG, O’Donnell M, Kuriyan J (2006) Crystal structure of the catalytic α subunit of E. coli replicative DNA polymerase III. Cell 126:881–892.  https://doi.org/10.1016/j.cell.2006.07.028 CrossRefGoogle Scholar
  12. López De Saro FJ, Georgescu RE, O’Donnell MM (2003) A peptide switch regulates DNA polymerase processivity. Proc Natl Acad Sci USA 100:14689–14694.  https://doi.org/10.1073/pnas.2435454100 CrossRefGoogle Scholar
  13. Modrich P (2016) Mechanisms in E. coli and human mismatch repair. Nobel Lect 55:8490–8501.  https://doi.org/10.1002/anie.201601412 Google Scholar
  14. Nanfara MT, Babu VMP, Ghazy MA, Sutton MD (2016) Identification of β clamp–DNA interaction regions that impair the ability of E. coli to tolerate specific classes of DNA damage. PLoS One.  https://doi.org/10.1371/journal.pone.0163643 Google Scholar
  15. O’Donnell M, Kuriyan J, Kong XP, Stukenberg PT, Onrust R (1992) The sliding clamp of DNA polymerase III holoenzyme encircles DNA. Mol Biol Cell 3:953–957.  https://doi.org/10.1091/mbc.3.9.953 CrossRefGoogle Scholar
  16. Pandey P, Tarique KF, Mazumder M, Rehman SAA, Kumari N, Gourinath S (2016) Structural insight into β-Clamp and its interaction with DNA Ligase in Helicobacter pylori. Sci Rep.  https://doi.org/10.1038/srep31181 Google Scholar
  17. Patoli AA, Winter JA, Bunting KA (2013) The UmuC subunit of the E. coli DNA polymerase v shows a unique interaction with the β-clamp processivity factor. BMC Struct Biol.  https://doi.org/10.1186/1472-6807-13-12 Google Scholar
  18. Rothwell PJ, Waksman G (2005) Structure and mechanism of DNA polymerases. Adv Protein Chem 71:401–440.  https://doi.org/10.1016/S0065-3233(04)71011-6 CrossRefGoogle Scholar
  19. Theobald DL, Mitton-Fry RM, Wuttke DS (2003) Nucleic acid recognition by OB-fold proteins. Annu Rev Biophys Biomol Struct 32:115–133.  https://doi.org/10.1146/annurev.biophys.32.110601.142506 CrossRefGoogle Scholar
  20. Welch MM, McHenry CS (1982) Cloning and identification of the product of the dnaE gene of Escherichia coli. J Bacteriol 152:351–356Google Scholar

Copyright information

© The Genetics Society of Korea 2019

Authors and Affiliations

  1. 1.School of Biology, Queens Medical CentreUniversity of NottinghamNottinghamUK
  2. 2.Institute of MicrobiologyUniversity of SindhJamshoroPakistan

Personalised recommendations