Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Sep;175(18):6018–6027. doi: 10.1128/jb.175.18.6018-6027.1993

The Escherichia coli DNA polymerase III holoenzyme contains both products of the dnaX gene, tau and gamma, but only tau is essential.

A Blinkova 1, C Hervas 1, P T Stukenberg 1, R Onrust 1, M E O'Donnell 1, J R Walker 1
PMCID: PMC206684  PMID: 8376347

Abstract

The replicative polymerase of Escherichia coli, DNA polymerase III, consists of a three-subunit core polymerase plus seven accessory subunits. Of these seven, tau and gamma are products of one replication gene, dnaX. The shorter gamma is created from within the tau reading frame by a programmed ribosomal -1 frameshift over codons 428 and 429 followed by a stop codon in the new frame. Two temperature-sensitive mutations are available in dnaX. The 2016(Ts) mutation altered both tau and gamma by changing codon 118 from glycine to aspartate; the 36(Ts) mutation affected the activity only of tau because it altered codon 601 (from glutamate to lysine). Evidence which indicates that, of these two proteins, only the longer tau is essential includes the following. (i) The 36(Ts) mutation is a temperature-sensitive lethal allele, and overproduction of wild-type gamma cannot restore its growth. (ii) An allele which produced tau only could be substituted for the wild-type chromosomal gene, but a gamma-only allele could not substitute for the wild-type dnaX in the haploid state. Thus, the shorter subunit gamma is not essential, suggesting that tau can be substitute for the usual function(s) of gamma. Consistent with these results, we found that a functional polymerase was assembled from nine pure subunits in the absence of the gamma subunit. However, the possibility that, in cells growing without gamma, proteolysis of tau to form a gamma-like product in amounts below the Western blot (immunoblot) sensitivity level cannot be excluded.

Full text

PDF
6019

Images in this article

6023Image
on p.6023
6024Image
on p.6024
6024Image
on p.6024

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Biswas S. B., Kornberg A. Nucleoside triphosphate binding to DNA polymerase III holoenzyme of Escherichia coli. A direct photoaffinity labeling study. J Biol Chem. 1984 Jun 25;259(12):7990–7993. [PubMed] [Google Scholar]
  2. Blinkowa A. L., Walker J. R. Programmed ribosomal frameshifting generates the Escherichia coli DNA polymerase III gamma subunit from within the tau subunit reading frame. Nucleic Acids Res. 1990 Apr 11;18(7):1725–1729. doi: 10.1093/nar/18.7.1725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bonner C. A., Stukenberg P. T., Rajagopalan M., Eritja R., O'Donnell M., McEntee K., Echols H., Goodman M. F. Processive DNA synthesis by DNA polymerase II mediated by DNA polymerase III accessory proteins. J Biol Chem. 1992 Jun 5;267(16):11431–11438. [PubMed] [Google Scholar]
  4. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  5. Chen E. Y., Seeburg P. H. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA. 1985 Apr;4(2):165–170. doi: 10.1089/dna.1985.4.165. [DOI] [PubMed] [Google Scholar]
  6. Filip C. C., Allen J. S., Gustafson R. A., Allen R. G., Walker J. R. Bacterial cell division regulation: characterization of the dnaH locus of Escherichia coli. J Bacteriol. 1974 Aug;119(2):443–449. doi: 10.1128/jb.119.2.443-449.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Flower A. M., McHenry C. S. The adjacent dnaZ and dnaX genes of Escherichia coli are contained within one continuous open reading frame. Nucleic Acids Res. 1986 Oct 24;14(20):8091–8101. doi: 10.1093/nar/14.20.8091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Flower A. M., McHenry C. S. The gamma subunit of DNA polymerase III holoenzyme of Escherichia coli is produced by ribosomal frameshifting. Proc Natl Acad Sci U S A. 1990 May;87(10):3713–3717. doi: 10.1073/pnas.87.10.3713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fradkin L. G., Kornberg A. Prereplicative complexes of components of DNA polymerase III holoenzyme of Escherichia coli. J Biol Chem. 1992 May 25;267(15):10318–10322. [PubMed] [Google Scholar]
  10. Griep M. A., McHenry C. S. Fluorescence energy transfer between the primer and the beta subunit of the DNA polymerase III holoenzyme. J Biol Chem. 1992 Feb 15;267(5):3052–3059. [PubMed] [Google Scholar]
  11. HOWARD-FLANDERS P., SIMSON E., THERIOT L. A LOCUS THAT CONTROLS FILAMENT FORMATION AND SENSITIVITY TO RADIATION IN ESCHERICHIA COLI K-12. Genetics. 1964 Feb;49:237–246. doi: 10.1093/genetics/49.2.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hamilton C. M., Aldea M., Washburn B. K., Babitzke P., Kushner S. R. New method for generating deletions and gene replacements in Escherichia coli. J Bacteriol. 1989 Sep;171(9):4617–4622. doi: 10.1128/jb.171.9.4617-4622.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hawker J. R., Jr, McHenry C. S. Monoclonal antibodies specific for the tau subunit of the DNA polymerase III holoenzyme of Escherichia coli. Use to demonstrate that tau is the product of the dnaZX gene and that both it and gamma, the dnaZ gene product, are integral components of the same enzyme assembly. J Biol Chem. 1987 Sep 15;262(26):12722–12727. [PubMed] [Google Scholar]
  14. Henson J. M., Chu H., Irwin C. A., Walker J. R. Isolation and characterization of dnaX and dnaY temperature-sensitive mutants of Escherichia coli. Genetics. 1979 Aug;92(4):1041–1059. doi: 10.1093/genetics/92.4.1041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hübscher U., Kornberg A. The dnaZ protein, the gamma subunit of DNA polymerase III holoenzyme of Escherichia coli. J Biol Chem. 1980 Dec 25;255(24):11698–11703. [PubMed] [Google Scholar]
  16. Johanson K. O., McHenry C. S. Adenosine 5'-O-(3-thiotriphosphate) can support the formation of an initiation complex between the DNA polymerase III holoenzyme and primed DNA. J Biol Chem. 1984 Apr 10;259(7):4589–4595. [PubMed] [Google Scholar]
  17. Kodaira M., Biswas S. B., Kornberg A. The dnaX gene encodes the DNA polymerase III holoenzyme tau subunit, precursor of the gamma subunit, the dnaZ gene product. Mol Gen Genet. 1983;192(1-2):80–86. doi: 10.1007/BF00327650. [DOI] [PubMed] [Google Scholar]
  18. Kong X. P., Onrust R., O'Donnell M., Kuriyan J. Three-dimensional structure of the beta subunit of E. coli DNA polymerase III holoenzyme: a sliding DNA clamp. Cell. 1992 May 1;69(3):425–437. doi: 10.1016/0092-8674(92)90445-i. [DOI] [PubMed] [Google Scholar]
  19. Lederberg E. M., Cohen S. N. Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid. J Bacteriol. 1974 Sep;119(3):1072–1074. doi: 10.1128/jb.119.3.1072-1074.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lee S. H., Kanda P., Kennedy R. C., Walker J. R. Relation of the Escherichia coli dnaX gene to its two products--the tau and gamma subunits of DNA polymerase III holoenzyme. Nucleic Acids Res. 1987 Oct 12;15(19):7663–7675. doi: 10.1093/nar/15.19.7663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lee S. H., Walker J. R. Escherichia coli DnaX product, the tau subunit of DNA polymerase III, is a multifunctional protein with single-stranded DNA-dependent ATPase activity. Proc Natl Acad Sci U S A. 1987 May;84(9):2713–2717. doi: 10.1073/pnas.84.9.2713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lerner C. G., Inouye M. Low copy number plasmids for regulated low-level expression of cloned genes in Escherichia coli with blue/white insert screening capability. Nucleic Acids Res. 1990 Aug 11;18(15):4631–4631. doi: 10.1093/nar/18.15.4631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Maki H., Maki S., Kornberg A. DNA Polymerase III holoenzyme of Escherichia coli. IV. The holoenzyme is an asymmetric dimer with twin active sites. J Biol Chem. 1988 May 15;263(14):6570–6578. [PubMed] [Google Scholar]
  25. Maki S., Kornberg A. DNA polymerase III holoenzyme of Escherichia coli. I. Purification and distinctive functions of subunits tau and gamma, the dnaZX gene products. J Biol Chem. 1988 May 15;263(14):6547–6554. [PubMed] [Google Scholar]
  26. Maki S., Kornberg A. DNA polymerase III holoenzyme of Escherichia coli. II. A novel complex including the gamma subunit essential for processive synthesis. J Biol Chem. 1988 May 15;263(14):6555–6560. [PubMed] [Google Scholar]
  27. Maki S., Kornberg A. DNA polymerase III holoenzyme of Escherichia coli. III. Distinctive processive polymerases reconstituted from purified subunits. J Biol Chem. 1988 May 15;263(14):6561–6569. [PubMed] [Google Scholar]
  28. Marians K. J. Prokaryotic DNA replication. Annu Rev Biochem. 1992;61:673–719. doi: 10.1146/annurev.bi.61.070192.003325. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. McHenry C. S. Purification and characterization of DNA polymerase III'. Identification of tau as a subunit of the DNA polymerase III holoenzyme. J Biol Chem. 1982 Mar 10;257(5):2657–2663. [PubMed] [Google Scholar]
  31. McHenry C., Kornberg A. DNA polymerase III holoenzyme of Escherichia coli. Purification and resolution into subunits. J Biol Chem. 1977 Sep 25;252(18):6478–6484. [PubMed] [Google Scholar]
  32. Mullin D. A., Woldringh C. L., Henson J. M., Walker J. R. Cloning of the Escherichia coli dnaZX region and identification of its products. Mol Gen Genet. 1983;192(1-2):73–79. doi: 10.1007/BF00327649. [DOI] [PubMed] [Google Scholar]
  33. O'Donnell M. E. Accessory proteins bind a primed template and mediate rapid cycling of DNA polymerase III holoenzyme from Escherichia coli. J Biol Chem. 1987 Dec 5;262(34):16558–16565. [PubMed] [Google Scholar]
  34. O'Donnell M., Studwell P. S. Total reconstitution of DNA polymerase III holoenzyme reveals dual accessory protein clamps. J Biol Chem. 1990 Jan 15;265(2):1179–1187. [PubMed] [Google Scholar]
  35. Onrust R., O'Donnell M. DNA polymerase III accessory proteins. II. Characterization of delta and delta'. J Biol Chem. 1993 Jun 5;268(16):11766–11772. [PubMed] [Google Scholar]
  36. Onrust R., Stukenberg P. T., O'Donnell M. Analysis of the ATPase subassembly which initiates processive DNA synthesis by DNA polymerase III holoenzyme. J Biol Chem. 1991 Nov 15;266(32):21681–21686. [PubMed] [Google Scholar]
  37. Park C., Hazelbauer G. L. Transfer of chromosomal mutations to plasmids via Hfr-mediated conduction. J Bacteriol. 1986 Jan;165(1):312–314. doi: 10.1128/jb.165.1.312-314.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Scheuermann R. H., Echols H. A separate editing exonuclease for DNA replication: the epsilon subunit of Escherichia coli DNA polymerase III holoenzyme. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7747–7751. doi: 10.1073/pnas.81.24.7747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sinha N. K., Morris C. F., Alberts B. M. Efficient in vitro replication of double-stranded DNA templates by a purified T4 bacteriophage replication system. J Biol Chem. 1980 May 10;255(9):4290–4293. [PubMed] [Google Scholar]
  41. Spanos A., Sedgwick S. G., Yarranton G. T., Hübscher U., Banks G. R. Detection of the catalytic activities of DNA polymerases and their associated exonucleases following SDS-polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Apr 24;9(8):1825–1839. doi: 10.1093/nar/9.8.1825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. Stukenberg P. T., Studwell-Vaughan P. S., O'Donnell M. Mechanism of the sliding beta-clamp of DNA polymerase III holoenzyme. J Biol Chem. 1991 Jun 15;266(17):11328–11334. [PubMed] [Google Scholar]
  45. Tsuchihashi Z., Brown P. O. Sequence requirements for efficient translational frameshifting in the Escherichia coli dnaX gene and the role of an unstable interaction between tRNA(Lys) and an AAG lysine codon. Genes Dev. 1992 Mar;6(3):511–519. doi: 10.1101/gad.6.3.511. [DOI] [PubMed] [Google Scholar]
  46. Tsuchihashi Z., Kornberg A. ATP interactions of the tau and gamma subunits of DNA polymerase III holoenzyme of Escherichia coli. J Biol Chem. 1989 Oct 25;264(30):17790–17795. [PubMed] [Google Scholar]
  47. Tsuchihashi Z., Kornberg A. Translational frameshifting generates the gamma subunit of DNA polymerase III holoenzyme. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2516–2520. doi: 10.1073/pnas.87.7.2516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tsuchihashi Z. Translational frameshifting in the Escherichia coli dnaX gene in vitro. Nucleic Acids Res. 1991 May 11;19(9):2457–2462. doi: 10.1093/nar/19.9.2457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wickner S., Hurwitz J. Involvement of escherichia coli dnaZ gene product in DNA elongation in vitro. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1053–1057. doi: 10.1073/pnas.73.4.1053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wickner S. Mechanism of DNA elongation catalyzed by Escherichia coli DNA polymerase III, dnaZ protein, and DNA elongation factors I and III. Proc Natl Acad Sci U S A. 1976 Oct;73(10):3511–3515. doi: 10.1073/pnas.73.10.3511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wickner W., Kornberg A. A holoenzyme form of deoxyribonucleic acid polymerase III. Isolation and properties. J Biol Chem. 1974 Oct 10;249(19):6244–6249. [PubMed] [Google Scholar]
  52. Wickner W., Schekman R., Geider K., Kornberg A. A new form of DNA polymerase 3 and a copolymerase replicate a long, single-stranded primer-template. Proc Natl Acad Sci U S A. 1973 Jun;70(6):1764–1767. doi: 10.1073/pnas.70.6.1764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Willetts N. S., Clark A. J., Low B. Genetic location of certain mutations conferring recombination deficiency in Escherichia coli. J Bacteriol. 1969 Jan;97(1):244–249. doi: 10.1128/jb.97.1.244-249.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wu C. A., Zechner E. L., Hughes A. J., Jr, Franden M. A., McHenry C. S., Marians K. J. Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. IV. Reconstitution of an asymmetric, dimeric DNA polymerase III holoenzyme. J Biol Chem. 1992 Feb 25;267(6):4064–4073. [PubMed] [Google Scholar]
  55. Xiao H., Dong Z., O'Donnell M. DNA polymerase III accessory proteins. IV. Characterization of chi and psi. J Biol Chem. 1993 Jun 5;268(16):11779–11784. [PubMed] [Google Scholar]
  56. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  57. Yeung T., Mullin D. A., Chen K. S., Craig E. A., Bardwell J. C., Walker J. R. Sequence and expression of the Escherichia coli recR locus. J Bacteriol. 1990 Oct;172(10):6042–6047. doi: 10.1128/jb.172.10.6042-6047.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Yin K. C., Blinkowa A., Walker J. R. Nucleotide sequence of the Escherichia coli replication gene dnaZX. Nucleic Acids Res. 1986 Aug 26;14(16):6541–6549. doi: 10.1093/nar/14.16.6541. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES