In:
Journal of Bacteriology, American Society for Microbiology, Vol. 187, No. 20 ( 2005-10-15), p. 7081-7089
Abstract:
The catalytic core of Escherichia coli DNA 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 recorded 1 H- 15 N 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.
Type of Medium:
Online Resource
ISSN:
0021-9193
,
1098-5530
DOI:
10.1128/JB.187.20.7081-7089.2005
Language:
English
Publisher:
American Society for Microbiology
Publication Date:
2005
detail.hit.zdb_id:
1481988-0
SSG:
12
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