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. 1983 Feb 25;11(4):987–997. doi: 10.1093/nar/11.4.987

The dnaC protein of Escherichia coli. Purification, physical properties and interaction with dnaB protein.

E Lanka, H Schuster
PMCID: PMC325772  PMID: 6298736

Abstract

E.coli dnaC protein was purified to near-homogeneity in using a dnaC complementation assay [S.Wickner, I.Berkower, M.Wright, and J.Hurwitz (1973) Proc. Natl. Acad. Sci. USA 70, 2369-2373]. Purification was achieved by taking advantage of the hydrophobic interaction of dnaC protein with aliphatic and aromatic matrixes and with Brij58 as stabilizing agent. A sedimentation coefficient for the dnaC protein of 2.6 S corresponding to a molecular weight of approximately 26,000 was estimated from glycerol gradient centrifugation. A polypeptide molecular weight of 28,000 was determined by densitometry on a denaturing gel. In the presence of ATP the dnaC protein forms a complex with dnaB protein [S.Wickner and J.Hurwitz (1975) Proc.Natl.Acad.Sci. USA 72, 921-925]. For the dnaB . dnaC complex a sedimentation coefficient of 14.5 S was measured by glycerol gradient centrifugation, indicating a molecular weight of about 400,000. The ratio of the dnaC and dnaB polypeptides in the complex is approximately 1, as determined on a denaturing gel. It is suggested that the complex consists of the dnaB protein hexamer and six dnaC polypeptides amounting to a calculated molecular weight of about 450,000.

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Selected References

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  1. Arai K., Kornberg A. A general priming system employing only dnaB protein and primase for DNA replication. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4308–4312. doi: 10.1073/pnas.76.9.4308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arai K., Yasuda S., Kornberg A. Mechanism of dnaB protein action. I. Crystallization and properties of dnaB protein, an essential replication protein in Escherichia coli. J Biol Chem. 1981 May 25;256(10):5247–5252. [PubMed] [Google Scholar]
  3. Bouché J. P., Zechel K., Kornberg A. dnaG gene product, a rifampicin-resistant RNA polymerase, initiates the conversion of a single-stranded coliphage DNA to its duplex replicative form. J Biol Chem. 1975 Aug 10;250(15):5995–6001. [PubMed] [Google Scholar]
  4. Collins J., Williams P., Helinski D. R. Plasmid ColE1 DNA replication in Escherichia coli strains temperature-sensitive for DNA replication. Mol Gen Genet. 1975;136(4):273–289. doi: 10.1007/BF00341713. [DOI] [PubMed] [Google Scholar]
  5. Edelbluth C., Lanka E., von der Hude W., Mikolajczyk M., Schuster H. Association of the prophage P1ban protein with the dnaB protein of Escherichia coli. Overproduction of ban protein by a P1bac crr mutant. Eur J Biochem. 1979 Mar;94(2):427–435. doi: 10.1111/j.1432-1033.1979.tb12910.x. [DOI] [PubMed] [Google Scholar]
  6. Gefter M. L., Hirota Y., Kornberg T., Wechsler J. A., Barnoux C. Analysis of DNA polymerases II and 3 in mutants of Escherichia coli thermosensitive for DNA synthesis. Proc Natl Acad Sci U S A. 1971 Dec;68(12):3150–3153. doi: 10.1073/pnas.68.12.3150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Günther E., Mikolajczyk M., Schuster H. Stabilization by ATP and ADP of Escherichia coli dnaB protein activity. J Biol Chem. 1981 Dec 10;256(23):11970–11973. [PubMed] [Google Scholar]
  8. Hasunuma K., Sekiguchi M. Effect of dna mutations on the replication of plasmid pSC101 in Escherichia coli K-12. J Bacteriol. 1979 Mar;137(3):1095–1099. doi: 10.1128/jb.137.3.1095-1099.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kranias E. G., Dumas L. B. Replication of bacteriophage phiX174 DNA in a temperature-sensitive dnaC mutant of escherichia coli C. J Virol. 1974 Jan;13(1):146–154. doi: 10.1128/jvi.13.1.146-154.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lanka E., Edelbluth C., Schlicht M., Schuster H. Escherichia coli dnaB protein. Affinity chromatography on immobilized nucleotides. J Biol Chem. 1978 Aug 25;253(16):5847–5851. [PubMed] [Google Scholar]
  11. Lanka E., Mikolajczyk M., Schlicht M., Schuster H. Association of the prophage P1ban protein with the dnaB protein of Escherichia coli. J Biol Chem. 1978 Jul 10;253(13):4746–4753. [PubMed] [Google Scholar]
  12. McMacken R., Kornberg A. A multienzyme system for priming the replication of phiX174 viral DNA. J Biol Chem. 1978 May 10;253(9):3313–3319. [PubMed] [Google Scholar]
  13. Reha-Krantz L. J., Hurwitz J. The dnaB gene product of Escherichia coli. I. Purification, homogeneity, and physical properties. J Biol Chem. 1978 Jun 10;253(11):4043–4050. [PubMed] [Google Scholar]
  14. Schekman R., Weiner J. H., Weiner A., Kornberg A. Ten proteins required for conversion of phiX174 single-stranded DNA to duplex form in vitro. Resolution and reconstitution. J Biol Chem. 1975 Aug 10;250(15):5859–5865. [PubMed] [Google Scholar]
  15. Schuster H., Mikolajczyk M., Rohrschneider J., Geschke B. phiX174 DNA-dependent DNA synthesis in vitro: requirement for P1 ban protein in dnaB mutant extracts of Escherichia coli. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3907–3911. doi: 10.1073/pnas.72.10.3907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Schuster H., Schlicht M., Lanka E., Mikolajczyk M., Edelbluth C. DNA synthesis in an Escherichia coli dna B dnaC mutant. Mol Gen Genet. 1977 Feb 28;151(1):11–16. doi: 10.1007/BF00446907. [DOI] [PubMed] [Google Scholar]
  17. Sclafani R. A., Wechsler J. A. Deoxyribonucleic acid initiation mutation dnaB252 is suppressed by elevated dnaC+ gene dosage. J Bacteriol. 1981 Apr;146(1):418–421. doi: 10.1128/jb.146.1.418-421.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Sclafani R. A., Wechsler J. A. Suppression of dnaC alleles by the dnaB analog (ban protein) of bacteriophage P1. J Bacteriol. 1981 Apr;146(1):321–324. doi: 10.1128/jb.146.1.321-324.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Staudenbauer W. L., Lanka E., Schuster H. Replication of small plasmids in extracts of Escherichia coli: involvement of the dnaB and dnaC protein in the replication of early replicative intermediates. Mol Gen Genet. 1978 Jul 4;162(3):243–249. doi: 10.1007/BF00268849. [DOI] [PubMed] [Google Scholar]
  20. Wechsler J. A. Genetic and phenotypic characterization of dnaC mutations. J Bacteriol. 1975 Feb;121(2):594–599. doi: 10.1128/jb.121.2.594-599.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Wechsler J. A., Gross J. D. Escherichia coli mutants temperature-sensitive for DNA synthesis. Mol Gen Genet. 1971;113(3):273–284. doi: 10.1007/BF00339547. [DOI] [PubMed] [Google Scholar]
  22. Wickner S. H. DNA replication proteins of Escherichia coli and phage lambda. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):303–310. doi: 10.1101/sqb.1979.043.01.037. [DOI] [PubMed] [Google Scholar]
  23. Wickner S. H. DNA replication proteins of Escherichia coli. Annu Rev Biochem. 1978;47:1163–1191. doi: 10.1146/annurev.bi.47.070178.005503. [DOI] [PubMed] [Google Scholar]
  24. Wickner S., Berkower I., Wright M., Hurwitz J. Studies on in vitro DNA synthesis: purification of dna C gene product containing dna D activity from Escherichia coli. Proc Natl Acad Sci U S A. 1973 Aug;70(8):2369–2373. doi: 10.1073/pnas.70.8.2369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wickner S., Hurwitz J. Interaction of Escherichia coli dnaB and dnaC(D) gene products in vitro. Proc Natl Acad Sci U S A. 1975 Mar;72(3):921–925. doi: 10.1073/pnas.72.3.921. [DOI] [PMC free article] [PubMed] [Google Scholar]

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