Nuclear Magnetic Resonance Solution Structure of theEscherichia coliDNA Polymerase III θ Subunit

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Abstract
The catalytic core ofEscherichia coliDNA polymerase III holoenzyme contains three subunits: α, ε, and θ. The α subunit contains the polymerase, and the ε subunit contains the exonucleolytic proofreading function. The small (8-kDa) θ subunit binds only to ε. Its function is not well understood, although it was shown to exert a small stabilizing effect on the ε proofreading function. In order to help elucidate its function, we undertook a determination of its solution structure. In aqueous solution, θ yielded poor-quality nuclear magnetic resonance spectra, presumably due to conformational exchange and/or protein aggregation. Based on our recently determined structure of the θ homolog from bacteriophage P1, named HOT, we constructed a homology model of θ. This model suggested that the unfavorable behavior of θ might arise from exposed hydrophobic residues, particularly toward the end of α-helix 3. In gel filtration studies, θ elutes later than expected, indicating that aggregation is potentially responsible for these problems. To address this issue, we recorded1H-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 θ in 60/40 (vol/vol) water-methanol is similar to that of HOT but differs significantly from a previously reported θ structure. The new θ structure is expected to provide additional insight into its physiological role and its effect on the ε proofreading subunit.