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Structure of the theta subunit of Escherichia coli DNA polymerase III in complex with the epsilon subunit.

Keniry MA, Park AY, Owen EA, Hamdan SM, Pintacuda G, Otting G, Dixon NE
J Bacteriol 188 4464-73 (2006)
Cited: 23 times
EuropePMC logo PMID: 16740953

Abstract

The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits, the alpha, epsilon, and theta subunits. The theta subunit is the smallest and least understood subunit. The three-dimensional structure of theta in a complex with the unlabeled N-terminal domain of the epsilon subunit, epsilon186, was determined by multidimensional nuclear magnetic resonance spectroscopy. The structure was refined using pseudocontact shifts that resulted from inserting a lanthanide ion (Dy3+, Er3+, or Ho3+) at the active site of epsilon186. The structure determination revealed a three-helix bundle fold that is similar to the solution structures of theta in a methanol-water buffer and of the bacteriophage P1 homolog, HOT, in aqueous buffer. Conserved nuclear Overhauser enhancement (NOE) patterns obtained for free and complexed theta show that most of the structure changes little upon complex formation. Discrepancies with respect to a previously published structure of free theta (Keniry et al., Protein Sci. 9:721-733, 2000) were attributed to errors in the latter structure. The present structure satisfies the pseudocontact shifts better than either the structure of theta in methanol-water buffer or the structure of HOT. satisfies these shifts. The epitope of epsilon186 on theta was mapped by NOE difference spectroscopy and was found to involve helix 1 and the C-terminal part of helix 3. The pseudocontact shifts indicated that the helices of theta are located about 15 A or farther from the lanthanide ion in the active site of epsilon186, in agreement with the extensive biochemical data for the theta-epsilon system.

Articles - 2axd mentioned but not cited (3)

  1. Protein-protein HADDocking using exclusively pseudocontact shifts. Schmitz C, Bonvin AM. J Biomol NMR 50 263-266 (2011)
  2. Structure of the theta subunit of Escherichia coli DNA polymerase III in complex with the epsilon subunit. Keniry MA, Park AY, Owen EA, Hamdan SM, Pintacuda G, Otting G, Dixon NE. J Bacteriol 188 4464-4473 (2006)
  3. Evidence for moonlighting functions of the θ subunit of Escherichia coli DNA polymerase III. Dietrich M, Pedró L, García J, Pons M, Hüttener M, Paytubi S, Madrid C, Juárez A. J Bacteriol 196 1102-1112 (2014)


Reviews citing this publication (10)

  1. Protein NMR using paramagnetic ions. Otting G. Annu Rev Biophys 39 387-405 (2010)
  2. DNA replicases from a bacterial perspective. McHenry CS. Annu Rev Biochem 80 403-436 (2011)
  3. Architecture and conservation of the bacterial DNA replication machinery, an underexploited drug target. Robinson A, Causer RJ, Dixon NE. Curr Drug Targets 13 352-372 (2012)
  4. Essential biological processes of an emerging pathogen: DNA replication, transcription, and cell division in Acinetobacter spp. Robinson A, Brzoska AJ, Turner KM, Withers R, Harry EJ, Lewis PJ, Dixon NE. Microbiol Mol Biol Rev 74 273-297 (2010)
  5. Structure determination of protein-protein complexes with long-range anisotropic paramagnetic NMR restraints. Hass MA, Ubbink M. Curr Opin Struct Biol 24 45-53 (2014)
  6. The structural analysis of protein-protein interactions by NMR spectroscopy. O'Connell MR, Gamsjaeger R, Mackay JP. Proteomics 9 5224-5232 (2009)
  7. NMR as a unique tool in assessment and complex determination of weak protein-protein interactions. Vinogradova O, Qin J. Top Curr Chem 326 35-45 (2012)
  8. A structural view of bacterial DNA replication. Oakley AJ. Protein Sci 28 990-1004 (2019)
  9. Protein-nucleic acid complexes and the role of mass spectrometry in their structure determination. Park AY, Robinson CV. Crit Rev Biochem Mol Biol 46 152-164 (2011)
  10. The Macromolecular Machines that Duplicate the Escherichia coli Chromosome as Targets for Drug Discovery. Kaguni JM. Antibiotics (Basel) 7 (2018)

Articles citing this publication (10)

  1. Numbat: an interactive software tool for fitting Deltachi-tensors to molecular coordinates using pseudocontact shifts. Schmitz C, Stanton-Cook MJ, Su XC, Otting G, Huber T. J Biomol NMR 41 179-189 (2008)
  2. Effect of macromolecular crowding on protein binding stability: modest stabilization and significant biological consequences. Batra J, Xu K, Qin S, Zhou HX. Biophys J 97 906-911 (2009)
  3. Structure of the Escherichia coli DNA polymerase III epsilon-HOT proofreading complex. Kirby TW, Harvey S, DeRose EF, Chalov S, Chikova AK, Perrino FW, Schaaper RM, London RE, Pedersen LC. J Biol Chem 281 38466-38471 (2006)
  4. Proofreading exonuclease on a tether: the complex between the E. coli DNA polymerase III subunits α, epsilon, θ and β reveals a highly flexible arrangement of the proofreading domain. Ozawa K, Horan NP, Robinson A, Yagi H, Hill FR, Jergic S, Xu ZQ, Loscha KV, Li N, Tehei M, Oakley AJ, Otting G, Huber T, Dixon NE. Nucleic Acids Res 41 5354-5367 (2013)
  5. Self-correcting mismatches during high-fidelity DNA replication. Fernandez-Leiro R, Conrad J, Yang JC, Freund SM, Scheres SH, Lamers MH. Nat Struct Mol Biol 24 140-143 (2017)
  6. The proofreading exonuclease subunit epsilon of Escherichia coli DNA polymerase III is tethered to the polymerase subunit alpha via a flexible linker. Ozawa K, Jergic S, Park AY, Dixon NE, Otting G. Nucleic Acids Res 36 5074-5082 (2008)
  7. Assignment of paramagnetic (15)N-HSQC spectra by heteronuclear exchange spectroscopy. John M, Headlam MJ, Dixon NE, Otting G. J Biomol NMR 37 43-51 (2007)
  8. An encodable lanthanide binding tag with reduced size and flexibility for measuring residual dipolar couplings and pseudocontact shifts in large proteins. Barb AW, Subedi GP. J Biomol NMR 64 75-85 (2016)
  9. Mutations that Separate the Functions of the Proofreading Subunit of the Escherichia coli Replicase. Whatley Z, Kreuzer KN. G3 (Bethesda) 5 1301-1311 (2015)
  10. DNA Polymerase α Subunit Residues and Interactions Required for Efficient Initiation Complex Formation Identified by a Genetic Selection. Lindow JC, Dohrmann PR, McHenry CS. J Biol Chem 290 16851-16860 (2015)


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