Structure of the θ subunit of Escherichia coli DNA polymerase III in complex with the ε subunit

Max A. Keniry*, Ah Young Park, Elisabeth A. Owen, Samir Hamdan, Guido Pintacuda, Gottfried Otting, Nicholas E. Dixon

*Corresponding author for this work

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23 Scopus citations

Abstract

The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits, the a, ε, and θ subunits. The θ subunit is the smallest and least understood subunit. The three-dimensional structure of θ in a complex with the unlabeled N-terminal domain of the ε subunit, ε 186, 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 ε 186. The structure determination revealed a three-helix bundle fold that is similar to the solution structures of θ 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 θ show that most of the structure changes little upon complex formation. Discrepancies with respect to a previously published structure of free θ (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 θ in methanol-water buffer or the structure of HOT. satisfies these shifts. The epitope of ε 186 on θ 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 θ are located about 15 Å or farther from the lanthanide ion in the active site of ε 186, in agreement with the extensive biochemical data for the θ-ε system.

Original languageEnglish (US)
Pages (from-to)4464-4473
Number of pages10
JournalJournal of Bacteriology
Volume188
Issue number12
DOIs
StatePublished - Jun 1 2006

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

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