Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Sep;175(17):5604–5610. doi: 10.1128/jb.175.17.5604-5610.1993

Identification, isolation, and overexpression of the gene encoding the psi subunit of DNA polymerase III holoenzyme.

J R Carter 1, M A Franden 1, R Aebersold 1, C S McHenry 1
PMCID: PMC206617  PMID: 8366044

Abstract

The gene encoding the psi subunit of DNA polymerase III holoenzyme, holD, was identified and isolated by an approach in which peptide sequence data were used to obtain a DNA hybridization probe. The gene, which maps to 99.3 centisomes, was sequenced and found to be identical to a previously uncharacterized open reading frame that overlaps the 5' end of rimI by 29 bases, contains 411 bp, and is predicted to encode a protein of 15,174 Da. When expressed in a plasmid that also expressed holC, holD directed expression of the psi subunit to about 3% of total soluble protein.

Full text

PDF
5607

Images in this article

5608Image
on p.5608

Selected References

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

  1. Aebersold R. H., Leavitt J., Saavedra R. A., Hood L. E., Kent S. B. Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. Proc Natl Acad Sci U S A. 1987 Oct;84(20):6970–6974. doi: 10.1073/pnas.84.20.6970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burgers P. M., Kornberg A. ATP activation of DNA polymerase III holoenzyme from Escherichia coli. II. Initiation complex: stoichiometry and reactivity. J Biol Chem. 1982 Oct 10;257(19):11474–11478. [PubMed] [Google Scholar]
  4. Burgers P. M., Kornberg A., Sakakibara Y. The dnaN gene codes for the beta subunit of DNA polymerase III holoenzyme of escherichia coli. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5391–5395. doi: 10.1073/pnas.78.9.5391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carter J. R., Franden M. A., Aebersold R., McHenry C. S. Identification, isolation, and characterization of the structural gene encoding the delta' subunit of Escherichia coli DNA polymerase III holoenzyme. J Bacteriol. 1993 Jun;175(12):3812–3822. doi: 10.1128/jb.175.12.3812-3822.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carter J. R., Franden M. A., Aebersold R., McHenry C. S. Molecular cloning, sequencing, and overexpression of the structural gene encoding the delta subunit of Escherichia coli DNA polymerase III holoenzyme. J Bacteriol. 1992 Nov;174(21):7013–7025. doi: 10.1128/jb.174.21.7013-7025.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Crute J. J., LaDuca R. J., Johanson K. O., McHenry C. S., Bambara R. A. Excess beta subunit can bypass the ATP requirement for highly processive synthesis by the Escherichia coli DNA polymerase III holoenzyme. J Biol Chem. 1983 Sep 25;258(18):11344–11349. [PubMed] [Google Scholar]
  8. DiFrancesco R., Bhatnagar S. K., Brown A., Bessman M. J. The interaction of DNA polymerase III and the product of the Escherichia coli mutator gene, mutD. J Biol Chem. 1984 May 10;259(9):5567–5573. [PubMed] [Google Scholar]
  9. Dong Z., Onrust R., Skangalis M., O'Donnell M. DNA polymerase III accessory proteins. I. holA and holB encoding delta and delta'. J Biol Chem. 1993 Jun 5;268(16):11758–11765. [PubMed] [Google Scholar]
  10. Fay P. J., Johanson K. O., McHenry C. S., Bambara R. A. Size classes of products synthesized processively by DNA polymerase III and DNA polymerase III holoenzyme of Escherichia coli. J Biol Chem. 1981 Jan 25;256(2):976–983. [PubMed] [Google Scholar]
  11. Fay P. J., Johanson K. O., McHenry C. S., Bambara R. A. Size classes of products synthesized processively by two subassemblies of Escherichia coli DNA polymerase III holoenzyme. J Biol Chem. 1982 May 25;257(10):5692–5699. [PubMed] [Google Scholar]
  12. 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]
  13. Fürste J. P., Pansegrau W., Frank R., Blöcker H., Scholz P., Bagdasarian M., Lanka E. Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector. Gene. 1986;48(1):119–131. doi: 10.1016/0378-1119(86)90358-6. [DOI] [PubMed] [Google Scholar]
  14. Geider K., Kornberg A. Conversion of the M13 viral single strand to the double-stranded replicative forms by purified proteins. J Biol Chem. 1974 Jul 10;249(13):3999–4005. [PubMed] [Google Scholar]
  15. Guyer M. S., Reed R. R., Steitz J. A., Low K. B. Identification of a sex-factor-affinity site in E. coli as gamma delta. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):135–140. doi: 10.1101/sqb.1981.045.01.022. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Heck J. D., Hatfield G. W. Valyl-tRNA synthetase gene of Escherichia coli K12. Molecular genetic characterization. J Biol Chem. 1988 Jan 15;263(2):857–867. [PubMed] [Google Scholar]
  18. 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]
  19. Kohara Y., Akiyama K., Isono K. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell. 1987 Jul 31;50(3):495–508. doi: 10.1016/0092-8674(87)90503-4. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Konigsberg W., Godson G. N. Evidence for use of rare codons in the dnaG gene and other regulatory genes of Escherichia coli. Proc Natl Acad Sci U S A. 1983 Feb;80(3):687–691. doi: 10.1073/pnas.80.3.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. 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]
  26. McHenry C. S., Crow W. DNA polymerase III of Escherichia coli. Purification and identification of subunits. J Biol Chem. 1979 Mar 10;254(5):1748–1753. [PubMed] [Google Scholar]
  27. McHenry C. S., Johanson K. O. DNA polymerase III holoenzyme of Escherichia coli: an asymmetric dimeric replicative complex containing distinguishable leading and lagging strand polymerases. Adv Exp Med Biol. 1984;179:315–319. doi: 10.1007/978-1-4684-8730-5_32. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. 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]
  31. 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]
  32. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
  34. Rudd K. E., Miller W., Ostell J., Benson D. A. Alignment of Escherichia coli K12 DNA sequences to a genomic restriction map. Nucleic Acids Res. 1990 Jan 25;18(2):313–321. doi: 10.1093/nar/18.2.313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. Washburn B. K., Kushner S. R. Construction and analysis of deletions in the structural gene (uvrD) for DNA helicase II of Escherichia coli. J Bacteriol. 1991 Apr;173(8):2569–2575. doi: 10.1128/jb.173.8.2569-2575.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Welch M. M., McHenry C. S. Cloning and identification of the product of the dnaE gene of Escherichia coli. J Bacteriol. 1982 Oct;152(1):351–356. doi: 10.1128/jb.152.1.351-356.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Wu C. A., Zechner E. L., Marians K. J. Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. I. Multiple effectors act to modulate Okazaki fragment size. J Biol Chem. 1992 Feb 25;267(6):4030–4044. [PubMed] [Google Scholar]
  44. Wu C. A., Zechner E. L., Reems J. A., McHenry C. S., Marians K. J. Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. V. Primase action regulates the cycle of Okazaki fragment synthesis. J Biol Chem. 1992 Feb 25;267(6):4074–4083. [PubMed] [Google Scholar]
  45. Xiao H., Crombie R., Dong Z., Onrust R., O'Donnell M. DNA polymerase III accessory proteins. III. holC and holD encoding chi and psi. J Biol Chem. 1993 Jun 5;268(16):11773–11778. [PubMed] [Google Scholar]
  46. Yoshikawa A., Isono S., Sheback A., Isono K. Cloning and nucleotide sequencing of the genes rimI and rimJ which encode enzymes acetylating ribosomal proteins S18 and S5 of Escherichia coli K12. Mol Gen Genet. 1987 Oct;209(3):481–488. doi: 10.1007/BF00331153. [DOI] [PubMed] [Google Scholar]
  47. Zechner E. L., Wu C. A., Marians K. J. Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. III. A polymerase-primase interaction governs primer size. J Biol Chem. 1992 Feb 25;267(6):4054–4063. [PubMed] [Google Scholar]

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

RESOURCES