Nuclear magnetic resonance solution structure of the Escherichia coli DNA polymerase III theta subunit.
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.
Mueller
Geoffrey A
GA
Laboratory of Structural Biology, MR-01, National Institute of Environmental Health Sciences, 111 Alexander Drive, Research Triangle Park, North Carolina 27709, USA.
Kirby
Thomas W
TW
DeRose
Eugene F
EF
Li
Dawei
D
Schaaper
Roel M
RM
London
Robert E
RE
D000438:Alcohols / Q000378:metabolism; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D004258:DNA Polymerase III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011522:Protons; D014764:Viral Proteins / Q000235:genetics
D000438:Alcohols; D029968:Escherichia coli Proteins; D011522:Protons; D014764:Viral Proteins; C500758:HOT protein, bacteriophage P1; C505238:holE protein, E coli; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase