A preliminary CD and NMR study of the Escherichia coli DNA polymerase III Theta subunit

The theta subunit of DNA polymerase III, the main replicative polymerase of Escherichia coli, has been examined by circular dichroism and by NMR spect roscopy, The polymerase core consists of three subunits: alpha, epsilon, an d theta, with alpha possessing the polymerase activity, epsilon functioning as a proofreading exonuclease, and theta, a small subunit of 8.9 kD, of un determined function. The theta subunit has been expressed in E. coli, and a CD analysis of theta indicates the presence of a significant amount of sec ondary structure: similar to 52% alpha helix, 9% beta sheet, 21% turns, and 18% random coil, However, at higher concentrations, theta yields a poorly- resolved 1D proton NMR spectrum in which both the amide protons and the met hyl protons show poor chemical shift dispersion. Subsequent H-1,N-15 HSQC a nalysis of uniformly-N-15-labeled theta supports the conclusion that approx imately half of the protein is reasonably well-structured. Another quarter of the protein, probably including some of the N-terminal region, is highly mobile, exhibiting a chemical shift pattern indicative of random coil stru cture. The remaining amide resonances exhibit significant broadening, indic ative of intermolecular and/or intramolecular exchange processes. Improved chemical shift dispersion and greater uniformity of resonance intensities i n the H-1-N-15 HSQC spectra resulted when [U-N-15]-theta was examined in th e presence of epsilon 186-the N-terminal domain of the epsilon-subunit, Fur ther work is currently in progress to define the solution structure of thet a and the theta-epsilon 186 complex. Proteins 1999; 36:111-116. Published 1 999 Wiley-Liss, Inc.dagger