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. 2000 Apr;9(4):721–733. doi: 10.1110/ps.9.4.721

NMR solution structure of the theta subunit of DNA polymerase III from Escherichia coli.

M A Keniry 1, H A Berthon 1, J Y Yang 1, C S Miles 1, N E Dixon 1
PMCID: PMC2144624  PMID: 10794414

Abstract

The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits (alpha, epsilon, and theta). The theta subunit is the smallest, but the least understood of the three. As a first step in a program aimed at understanding its function, the structure of the theta subunit has been determined by triple-resonance multidimensional NMR spectroscopy. Although only a small protein, theta was difficult to assign fully because approximately one-third of the protein is unstructured, and some sections of the remaining structured parts undergo intermediate intramolecular exchange. The secondary structure was deduced from the characteristic nuclear Overhauser effect patterns, the 3J(HN alpha) coupling constants and the consensus chemical shift index. The C-terminal third of the protein, which has many charged and hydrophilic amino acid residues, has no well-defined secondary structure and exists in a highly dynamic state. The N-terminal two-thirds has three helical segments (Gln10-Asp19, Glu38-Glu43, and His47-Glu54), one short extended segment (Pro34-Ala37), and a long loop (Ala20-Glu29), of which part may undergo intermediate conformational exchange. Solution of the three-dimensional structure by NMR techniques revealed that the helices fold in such a way that the surface of theta is bipolar, with one face of the protein containing most of the acidic residues and the other face containing most of the long chain basic residues. Preliminary chemical shift mapping experiments with a domain of the epsilon subunit have identified a loop region (Ala20-Glu29) in theta as the site of association with epsilon.

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Selected References

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  1. Barbato G., Ikura M., Kay L. E., Pastor R. W., Bax A. Backbone dynamics of calmodulin studied by 15N relaxation using inverse detected two-dimensional NMR spectroscopy: the central helix is flexible. Biochemistry. 1992 Jun 16;31(23):5269–5278. doi: 10.1021/bi00138a005. [DOI] [PubMed] [Google Scholar]
  2. Damberger F. F., Pelton J. G., Harrison C. J., Nelson H. C., Wemmer D. E. Solution structure of the DNA-binding domain of the heat shock transcription factor determined by multidimensional heteronuclear magnetic resonance spectroscopy. Protein Sci. 1994 Oct;3(10):1806–1821. doi: 10.1002/pro.5560031020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Farrow N. A., Muhandiram R., Singer A. U., Pascal S. M., Kay C. M., Gish G., Shoelson S. E., Pawson T., Forman-Kay J. D., Kay L. E. Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation. Biochemistry. 1994 May 17;33(19):5984–6003. doi: 10.1021/bi00185a040. [DOI] [PubMed] [Google Scholar]
  4. Grasberger B. L., Gronenborn A. M., Clore G. M. Analysis of the backbone dynamics of interleukin-8 by 15N relaxation measurements. J Mol Biol. 1993 Mar 20;230(2):364–372. doi: 10.1006/jmbi.1993.1152. [DOI] [PubMed] [Google Scholar]
  5. Güntert P., Mumenthaler C., Wüthrich K. Torsion angle dynamics for NMR structure calculation with the new program DYANA. J Mol Biol. 1997 Oct 17;273(1):283–298. doi: 10.1006/jmbi.1997.1284. [DOI] [PubMed] [Google Scholar]
  6. Holm L., Sander C. Protein structure comparison by alignment of distance matrices. J Mol Biol. 1993 Sep 5;233(1):123–138. doi: 10.1006/jmbi.1993.1489. [DOI] [PubMed] [Google Scholar]
  7. Kelman Z., O'Donnell M. DNA replication: enzymology and mechanisms. Curr Opin Genet Dev. 1994 Apr;4(2):185–195. doi: 10.1016/s0959-437x(05)80044-9. [DOI] [PubMed] [Google Scholar]
  8. Kuboniwa H., Grzesiek S., Delaglio F., Bax A. Measurement of HN-H alpha J couplings in calcium-free calmodulin using new 2D and 3D water-flip-back methods. J Biomol NMR. 1994 Nov;4(6):871–878. doi: 10.1007/BF00398416. [DOI] [PubMed] [Google Scholar]
  9. Laskowski R. A., Rullmannn J. A., MacArthur M. W., Kaptein R., Thornton J. M. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR. 1996 Dec;8(4):477–486. doi: 10.1007/BF00228148. [DOI] [PubMed] [Google Scholar]
  10. Li D., Allen D. L., Harvey S., Perrino F. W., Schaaper R. M., London R. E. A preliminary CD and NMR study of the Escherichia coli DNA polymerase III theta subunit. Proteins. 1999 Jul 1;36(1):111–116. doi: 10.1002/(sici)1097-0134(19990701)36:1<111::aid-prot9>3.0.co;2-1. [DOI] [PubMed] [Google Scholar]
  11. Maki H., Horiuchi T., Kornberg A. The polymerase subunit of DNA polymerase III of Escherichia coli. I. Amplification of the dnaE gene product and polymerase activity of the alpha subunit. J Biol Chem. 1985 Oct 25;260(24):12982–12986. [PubMed] [Google Scholar]
  12. Maki H., Kornberg A. The polymerase subunit of DNA polymerase III of Escherichia coli. II. Purification of the alpha subunit, devoid of nuclease activities. J Biol Chem. 1985 Oct 25;260(24):12987–12992. [PubMed] [Google Scholar]
  13. Matsuo H., Kupce E., Li H., Wagner G. Increased sensitivity in HNCA and HN(CO)CA experiments by selective C beta decoupling. J Magn Reson B. 1996 Oct;113(1):91–96. doi: 10.1006/jmrb.1996.0161. [DOI] [PubMed] [Google Scholar]
  14. McHenry C. S. DNA polymerase III holoenzyme of Escherichia coli. Annu Rev Biochem. 1988;57:519–550. doi: 10.1146/annurev.bi.57.070188.002511. [DOI] [PubMed] [Google Scholar]
  15. McHenry C. S. DNA polymerase III holoenzyme. Components, structure, and mechanism of a true replicative complex. J Biol Chem. 1991 Oct 15;266(29):19127–19130. [PubMed] [Google Scholar]
  16. Miles C. S., Weigelt J., Stamford N. P., Dammerova N., Otting G., Dixon N. E. Precise limits of the N-terminal domain of DnaB helicase determined by NMR spectroscopy. Biochem Biophys Res Commun. 1997 Feb 3;231(1):126–130. doi: 10.1006/bbrc.1997.6059. [DOI] [PubMed] [Google Scholar]
  17. O'Donnell M. E., Kornberg A. Dynamics of DNA polymerase III holoenzyme of Escherichia coli in replication of a multiprimed template. J Biol Chem. 1985 Oct 15;260(23):12875–12883. [PubMed] [Google Scholar]
  18. Pelletier H., Sawaya M. R., Wolfle W., Wilson S. H., Kraut J. Crystal structures of human DNA polymerase beta complexed with DNA: implications for catalytic mechanism, processivity, and fidelity. Biochemistry. 1996 Oct 1;35(39):12742–12761. doi: 10.1021/bi952955d. [DOI] [PubMed] [Google Scholar]
  19. Provencher S. W., Glöckner J. Estimation of globular protein secondary structure from circular dichroism. Biochemistry. 1981 Jan 6;20(1):33–37. doi: 10.1021/bi00504a006. [DOI] [PubMed] [Google Scholar]
  20. Scheuermann R., Tam S., Burgers P. M., Lu C., Echols H. Identification of the epsilon-subunit of Escherichia coli DNA polymerase III holoenzyme as the dnaQ gene product: a fidelity subunit for DNA replication. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7085–7089. doi: 10.1073/pnas.80.23.7085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  22. Studwell-Vaughan P. S., O'Donnell M. Constitution of the twin polymerase of DNA polymerase III holoenzyme. J Biol Chem. 1991 Oct 15;266(29):19833–19841. [PubMed] [Google Scholar]
  23. Studwell-Vaughan P. S., O'Donnell M. DNA polymerase III accessory proteins. V. Theta encoded by holE. J Biol Chem. 1993 Jun 5;268(16):11785–11791. [PubMed] [Google Scholar]
  24. Wishart D. S., Bigam C. G., Holm A., Hodges R. S., Sykes B. D. 1H, 13C and 15N random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-neighbor effects. J Biomol NMR. 1995 Jan;5(1):67–81. doi: 10.1007/BF00227471. [DOI] [PubMed] [Google Scholar]
  25. Wishart D. S., Sykes B. D. Chemical shifts as a tool for structure determination. Methods Enzymol. 1994;239:363–392. doi: 10.1016/s0076-6879(94)39014-2. [DOI] [PubMed] [Google Scholar]
  26. Wishart D. S., Sykes B. D. The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data. J Biomol NMR. 1994 Mar;4(2):171–180. doi: 10.1007/BF00175245. [DOI] [PubMed] [Google Scholar]
  27. Zhang O., Kay L. E., Olivier J. P., Forman-Kay J. D. Backbone 1H and 15N resonance assignments of the N-terminal SH3 domain of drk in folded and unfolded states using enhanced-sensitivity pulsed field gradient NMR techniques. J Biomol NMR. 1994 Nov;4(6):845–858. doi: 10.1007/BF00398413. [DOI] [PubMed] [Google Scholar]

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