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Journal of Bacteriology, June 2006, p. 4464-4473, Vol. 188, No. 12
0021-9193/06/$08.00+0     doi:10.1128/JB.01992-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Structure of the Subunit of Escherichia coli DNA Polymerase III in Complex with the Subunit

Max A. Keniry,^1* Ah Young Park,¹ Elisabeth A. Owen,¹ Samir M. Hamdan,¹ Guido Pintacuda,^1,2 Gottfried Otting,¹ and Nicholas E. Dixon¹

Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia,¹ Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden²

Received 29 December 2005/ Accepted 23 March 2006

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

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* Corresponding author. Mailing address: Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia. Phone: 61-2-61252863. Fax: 61-2-61250750. E-mail: max{at}rsc.anu.edu.au.

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Journal of Bacteriology, June 2006, p. 4464-4473, Vol. 188, No. 12
0021-9193/06/$08.00+0     doi:10.1128/JB.01992-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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