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Journal of Bacteriology, October 2005, p. 7081-7089, Vol. 187, No. 20
0021-9193/05/$08.00+0     doi:10.1128/JB.187.20.7081-7089.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Nuclear Magnetic Resonance Solution Structure of the Escherichia coli DNA Polymerase III {theta} Subunit{dagger}

Geoffrey A. Mueller,1 Thomas W. Kirby,1 Eugene F. DeRose,1 Dawei Li,1 Roel M. Schaaper,2 and Robert E. London1*

Laboratory of Structural Biology,1 Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Box 12233, Research Triangle Park, North Carolina 277092

Received 7 April 2005/ Accepted 20 July 2005

The catalytic core of Escherichia coli DNA polymerase III holoenzyme contains three subunits: {alpha}, {epsilon}, and {theta}. The {alpha} subunit contains the polymerase, and the {epsilon} subunit contains the exonucleolytic proofreading function. The small (8-kDa) {theta} subunit binds only to {epsilon}. Its function is not well understood, although it was shown to exert a small stabilizing effect on the {epsilon} proofreading function. In order to help elucidate its function, we undertook a determination of its solution structure. In aqueous solution, {theta} yielded poor-quality nuclear magnetic resonance spectra, presumably due to conformational exchange and/or protein aggregation. Based on our recently determined structure of the {theta} homolog from bacteriophage P1, named HOT, we constructed a homology model of {theta}. This model suggested that the unfavorable behavior of {theta} might arise from exposed hydrophobic residues, particularly toward the end of {alpha}-helix 3. In gel filtration studies, {theta} elutes later than expected, indicating that aggregation is potentially responsible for these problems. To address this issue, we recorded 1H-15N heteronuclear single quantum correlation (HSQC) spectra in water-alcohol mixed solvents and observed substantially improved dispersion and uniformity of peak intensities, facilitating a structural determination under these conditions. The structure of {theta} in 60/40 (vol/vol) water-methanol is similar to that of HOT but differs significantly from a previously reported {theta} structure. The new {theta} structure is expected to provide additional insight into its physiological role and its effect on the {epsilon} proofreading subunit.

* Corresponding author. Mailing address: MR-01, National Institute of Environmental Health Sciences, 111 Alexander Drive, Research Triangle Park, NC 27709. Phone: (919) 541-4879. Fax: (919) 541-5707. E-mail: london{at}niehs.nih.gov.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, October 2005, p. 7081-7089, Vol. 187, No. 20
0021-9193/05/$08.00+0     doi:10.1128/JB.187.20.7081-7089.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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